Novel mechanisms of MITF regulation and melanoma predisposition identified in a mouse suppressor screen

Abstract

MITF, a basic-Helix-Loop-Helix Zipper (bHLHZip) transcription factor, plays vital roles in melanocyte development and functions as an oncogene. To explore MITF regulation and its role in melanoma, we conducted a genetic screen for suppressors of the Mitf-associated pigmentation phenotype. An intragenic Mitf mutation was identified, leading to termination of MITF at the K316 SUMOylation site and loss of the C-end intrinsically disordered region (IDR). The resulting protein is more nuclear but less stable than wild-type MITF and retains DNA-binding ability. Interestingly, as a dimer, it can translocate wild-type and mutant MITF partners into the nucleus, improving its own stability and ensuring an active nuclear MITF supply. Interactions between K316 SUMOylation and S409 phosphorylation sites across monomers largely explain the observed effects. Notably, the recurrent melanoma-associated E318K mutation in MITF, which affects K316 SUMOylation, also alters protein regulation in concert with S409, unraveling a novel regulatory mechanism with unexpected disease insights.

Keywords: Mitf; melanoma; nuclear export; protein stability; suppressor; transcription.

Figure 1:. Coat color phenotypes and molecular alteration associated with the induced Mitf^mi-sp# suppressor mutation (Mitf^mi-sl)

(A) Graphical depiction of the MITF-WT, MITF-sp, and MITF-sl (MITF-sp#) proteins indicating the domains affected. Also shown are the post-translational modifications that have been reported in MITF. (B) NAW-Mitf^mi-ew/B6-Mitf^mi-sp and NAW-Mitf^mi-ew/B6-Mitf^mi-sp# compound heterozygotes. (C) B6-Mitf^Mi-Wh/B6-Mitf^mi-sp and B6-Mitf^Mi-Wh/B6-Mitf^mi-sp# compound heterozygotes. (D) B6-Mitf^mi-sp/B6-Mitf^mi and B6-Mitf^mi-sp#/B6-Mitf^mi compound heterozygotes. Notice the dramatic suppression of the phenotype from near-white to black coat color. (E) B6-Mitf^mi-sp/B6-Mitf^mi-rw and B6-Mitf^mi-sp#/B6-Mitf^mi-rw animals. (F) B6-Mitfmi-sp/B6-Mitfmi-vga9. (G) B6-Mitfmi-sp#/B6-Mitfmi-vga9. (H) B6-Mitfmi-sp/Mitfmi-sp. (I) B6-Mitfmi-sp #/Mitfmi-sp#. (J) Graphical depiction of the Mitf^mi-sl mutation. (K) Antibody staining of melanocytes and eye sections from Mitf^mi-sp and Mitf^mi-sl tissues. The antibodies are 6A5, which recognizes the C-end of MITF, and a polyclonal rabbit anti-MITF antibody. (L) DNA binding curves of recombinantly expressed human MITF-WT, MITF-sp, and MITF-sl proteins to M-box probe measured by Fluorescence anisotropy at 165 mM KCl and 300 mM KCl. MITF-WT protein in light and dark blue circles, MITF-sp light and dark green boxes, and MITF-sl in yellow and red triangles. Error bars represent two standard deviations of fit error at each point.

Figure 1:. Coat color phenotypes and molecular alteration associated with the induced Mitf^mi-sp# suppressor mutation (Mitf^mi-sl)

(A) Graphical depiction of the MITF-WT, MITF-sp, and MITF-sl (MITF-sp#) proteins indicating the domains affected. Also shown are the post-translational modifications that have been reported in MITF. (B) NAW-Mitf^mi-ew/B6-Mitf^mi-sp and NAW-Mitf^mi-ew/B6-Mitf^mi-sp# compound heterozygotes. (C) B6-Mitf^Mi-Wh/B6-Mitf^mi-sp and B6-Mitf^Mi-Wh/B6-Mitf^mi-sp# compound heterozygotes. (D) B6-Mitf^mi-sp/B6-Mitf^mi and B6-Mitf^mi-sp#/B6-Mitf^mi compound heterozygotes. Notice the dramatic suppression of the phenotype from near-white to black coat color. (E) B6-Mitf^mi-sp/B6-Mitf^mi-rw and B6-Mitf^mi-sp#/B6-Mitf^mi-rw animals. (F) B6-Mitfmi-sp/B6-Mitfmi-vga9. (G) B6-Mitfmi-sp#/B6-Mitfmi-vga9. (H) B6-Mitfmi-sp/Mitfmi-sp. (I) B6-Mitfmi-sp #/Mitfmi-sp#. (J) Graphical depiction of the Mitf^mi-sl mutation. (K) Antibody staining of melanocytes and eye sections from Mitf^mi-sp and Mitf^mi-sl tissues. The antibodies are 6A5, which recognizes the C-end of MITF, and a polyclonal rabbit anti-MITF antibody. (L) DNA binding curves of recombinantly expressed human MITF-WT, MITF-sp, and MITF-sl proteins to M-box probe measured by Fluorescence anisotropy at 165 mM KCl and 300 mM KCl. MITF-WT protein in light and dark blue circles, MITF-sp light and dark green boxes, and MITF-sl in yellow and red triangles. Error bars represent two standard deviations of fit error at each point.

Figure 1:. Coat color phenotypes and molecular alteration associated with the induced Mitf^mi-sp# suppressor mutation (Mitf^mi-sl)

(A) Graphical depiction of the MITF-WT, MITF-sp, and MITF-sl (MITF-sp#) proteins indicating the domains affected. Also shown are the post-translational modifications that have been reported in MITF. (B) NAW-Mitf^mi-ew/B6-Mitf^mi-sp and NAW-Mitf^mi-ew/B6-Mitf^mi-sp# compound heterozygotes. (C) B6-Mitf^Mi-Wh/B6-Mitf^mi-sp and B6-Mitf^Mi-Wh/B6-Mitf^mi-sp# compound heterozygotes. (D) B6-Mitf^mi-sp/B6-Mitf^mi and B6-Mitf^mi-sp#/B6-Mitf^mi compound heterozygotes. Notice the dramatic suppression of the phenotype from near-white to black coat color. (E) B6-Mitf^mi-sp/B6-Mitf^mi-rw and B6-Mitf^mi-sp#/B6-Mitf^mi-rw animals. (F) B6-Mitfmi-sp/B6-Mitfmi-vga9. (G) B6-Mitfmi-sp#/B6-Mitfmi-vga9. (H) B6-Mitfmi-sp/Mitfmi-sp. (I) B6-Mitfmi-sp #/Mitfmi-sp#. (J) Graphical depiction of the Mitf^mi-sl mutation. (K) Antibody staining of melanocytes and eye sections from Mitf^mi-sp and Mitf^mi-sl tissues. The antibodies are 6A5, which recognizes the C-end of MITF, and a polyclonal rabbit anti-MITF antibody. (L) DNA binding curves of recombinantly expressed human MITF-WT, MITF-sp, and MITF-sl proteins to M-box probe measured by Fluorescence anisotropy at 165 mM KCl and 300 mM KCl. MITF-WT protein in light and dark blue circles, MITF-sp light and dark green boxes, and MITF-sl in yellow and red triangles. Error bars represent two standard deviations of fit error at each point.

Figure 1:. Coat color phenotypes and molecular alteration associated with the induced Mitf^mi-sp# suppressor mutation (Mitf^mi-sl)

(A) Graphical depiction of the MITF-WT, MITF-sp, and MITF-sl (MITF-sp#) proteins indicating the domains affected. Also shown are the post-translational modifications that have been reported in MITF. (B) NAW-Mitf^mi-ew/B6-Mitf^mi-sp and NAW-Mitf^mi-ew/B6-Mitf^mi-sp# compound heterozygotes. (C) B6-Mitf^Mi-Wh/B6-Mitf^mi-sp and B6-Mitf^Mi-Wh/B6-Mitf^mi-sp# compound heterozygotes. (D) B6-Mitf^mi-sp/B6-Mitf^mi and B6-Mitf^mi-sp#/B6-Mitf^mi compound heterozygotes. Notice the dramatic suppression of the phenotype from near-white to black coat color. (E) B6-Mitf^mi-sp/B6-Mitf^mi-rw and B6-Mitf^mi-sp#/B6-Mitf^mi-rw animals. (F) B6-Mitfmi-sp/B6-Mitfmi-vga9. (G) B6-Mitfmi-sp#/B6-Mitfmi-vga9. (H) B6-Mitfmi-sp/Mitfmi-sp. (I) B6-Mitfmi-sp #/Mitfmi-sp#. (J) Graphical depiction of the Mitf^mi-sl mutation. (K) Antibody staining of melanocytes and eye sections from Mitf^mi-sp and Mitf^mi-sl tissues. The antibodies are 6A5, which recognizes the C-end of MITF, and a polyclonal rabbit anti-MITF antibody. (L) DNA binding curves of recombinantly expressed human MITF-WT, MITF-sp, and MITF-sl proteins to M-box probe measured by Fluorescence anisotropy at 165 mM KCl and 300 mM KCl. MITF-WT protein in light and dark blue circles, MITF-sp light and dark green boxes, and MITF-sl in yellow and red triangles. Error bars represent two standard deviations of fit error at each point.

Figure 2:. The carboxyl-domain of Mitf controls RNA and protein levels as well as its subcellular localization

(A) Western blot analysis of the Mitf-Flag proteins upon cycloheximide treatment. The dox-inducible A375P cells expressing the MITF-WT, MITF-sp, and MITF-sl proteins were treated with doxycycline for 24h to induce similar expression of the indicated mutant MITF proteins before treating them with 40 μg/ml cycloheximide (CHX) for 0, 1, 2, and 3 hours. The blots were stained using Flag antibody and protein quantitated using the odyssey imager and ImageJ. Actin was used as a loading control. (B), (C) Non-linear regression (one-phase decay) and half-life analysis of the indicated pS73- and S73-MITF proteins over time after CHX treatment in A375P melanoma cells. The relative MITF protein levels to T0 were calculated, and non-linear regression analysis was performed. Error bars represent SEM of at least three independent experiments. Statistically significant differences (Student’s t-test) are indicated by *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant. (D) Western blot analysis of subcellular fractions isolated from A375P melanoma cells induced for 24 hours to overexpress different MITF mutant proteins. MITF-WT, MITF-Wh, MITF-sp, and MITF-sl protein in whole cell lysate (W), cytoplasmic (C), and nuclear (N) fractions were visualized using FLAG antibody. Actin and γH2AX were loading controls for cytoplasmic and nuclear fractions, respectively. (E) Intensities of the indicated pS73- and S73-MITF protein bands in the cytoplasmic and nuclear fraction from the western blot analysis in (E) were quantified separately with ImageJ software and are depicted as percentages of the total amount of protein present in the two fractions. Error bars represent SEM of three independent experiments. Statistically significant differences (Student’s t-test) are indicated by * p< 0.05, *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant. (F) Western blot analysis of subcellular fractions isolated from A375P cells transiently co-overexpressing the MITF-sl protein with the MITF-Wh, MITF-mi, and MITF-ew mutant MITF proteins. MITF proteins in cytoplasmic (C) and nuclear (N) fractions were visualized using FLAG antibody. Actin and γH2AX were loading controls for cytoplasmic and nuclear fractions, respectively. The MITF-sl protein migrates as a doublet at 50–55 kDa, whereas the other mutants migrate at 65–70 kDa. (G) The intensities of the pS73- and S73- MITF-sl protein in the cytoplasmic and nuclear fractions from western blot analysis (G) were quantified separately with ImageJ software and are depicted as percentages of the total protein present in the two fractions. Error bars represent SEM of at least three independent experiments. Statistically significant differences (Student’s t-test) are indicated by * p< 0.05, *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant. (H) Quantification of band intensities of the pS73- and S73-versions of the MITF-Wh, MITF-mi, and MITF-ew proteins as determined from western blot analysis (G) in the nuclear fractions of A375P cells transiently co-overexpressing the MITF-sl protein with the indicated MITF mutant proteins. The intensities are depicted as percentages of the total amount of protein present in the two fractions. Error bars represent SEM of at least three independent experiments. Statistically significant differences (Student’s t-test) are indicated by * p< 0.05, *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant. (I), (J), and (K) Western blot analysis of the degradation of the MITF-sl protein in the presence of non-DNA binding MITF mutations (MITF-Wh, MITF-mi, and MITF-ew). The A375P cells were transiently co-transfected with MITF-sl and either MITF-mi, MITF-ew, or MITF-Wh for 24 hours before being treated with 55 μg/ml CHX. The amount of MITF protein was then compared by western blot using FLAG antibody. Actin was used as a loading control. The band intensities were quantified using ImageJ software. (L) and (M) Non-linear regression (one-phase decay) and half-life analysis of the indicated pS73- and S73-MITF proteins over time after CHX treatment. The relative MITF protein levels to T0 were calculated, and non-linear regression analysis was performed. Error bars represent SEM of at least three independent experiments. Statistically significant differences (Student’s t-test) are indicated by * p< 0.05, *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant.

Figure 2:. The carboxyl-domain of Mitf controls RNA and protein levels as well as its subcellular localization

(A) Western blot analysis of the Mitf-Flag proteins upon cycloheximide treatment. The dox-inducible A375P cells expressing the MITF-WT, MITF-sp, and MITF-sl proteins were treated with doxycycline for 24h to induce similar expression of the indicated mutant MITF proteins before treating them with 40 μg/ml cycloheximide (CHX) for 0, 1, 2, and 3 hours. The blots were stained using Flag antibody and protein quantitated using the odyssey imager and ImageJ. Actin was used as a loading control. (B), (C) Non-linear regression (one-phase decay) and half-life analysis of the indicated pS73- and S73-MITF proteins over time after CHX treatment in A375P melanoma cells. The relative MITF protein levels to T0 were calculated, and non-linear regression analysis was performed. Error bars represent SEM of at least three independent experiments. Statistically significant differences (Student’s t-test) are indicated by *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant. (D) Western blot analysis of subcellular fractions isolated from A375P melanoma cells induced for 24 hours to overexpress different MITF mutant proteins. MITF-WT, MITF-Wh, MITF-sp, and MITF-sl protein in whole cell lysate (W), cytoplasmic (C), and nuclear (N) fractions were visualized using FLAG antibody. Actin and γH2AX were loading controls for cytoplasmic and nuclear fractions, respectively. (E) Intensities of the indicated pS73- and S73-MITF protein bands in the cytoplasmic and nuclear fraction from the western blot analysis in (E) were quantified separately with ImageJ software and are depicted as percentages of the total amount of protein present in the two fractions. Error bars represent SEM of three independent experiments. Statistically significant differences (Student’s t-test) are indicated by * p< 0.05, *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant. (F) Western blot analysis of subcellular fractions isolated from A375P cells transiently co-overexpressing the MITF-sl protein with the MITF-Wh, MITF-mi, and MITF-ew mutant MITF proteins. MITF proteins in cytoplasmic (C) and nuclear (N) fractions were visualized using FLAG antibody. Actin and γH2AX were loading controls for cytoplasmic and nuclear fractions, respectively. The MITF-sl protein migrates as a doublet at 50–55 kDa, whereas the other mutants migrate at 65–70 kDa. (G) The intensities of the pS73- and S73- MITF-sl protein in the cytoplasmic and nuclear fractions from western blot analysis (G) were quantified separately with ImageJ software and are depicted as percentages of the total protein present in the two fractions. Error bars represent SEM of at least three independent experiments. Statistically significant differences (Student’s t-test) are indicated by * p< 0.05, *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant. (H) Quantification of band intensities of the pS73- and S73-versions of the MITF-Wh, MITF-mi, and MITF-ew proteins as determined from western blot analysis (G) in the nuclear fractions of A375P cells transiently co-overexpressing the MITF-sl protein with the indicated MITF mutant proteins. The intensities are depicted as percentages of the total amount of protein present in the two fractions. Error bars represent SEM of at least three independent experiments. Statistically significant differences (Student’s t-test) are indicated by * p< 0.05, *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant. (I), (J), and (K) Western blot analysis of the degradation of the MITF-sl protein in the presence of non-DNA binding MITF mutations (MITF-Wh, MITF-mi, and MITF-ew). The A375P cells were transiently co-transfected with MITF-sl and either MITF-mi, MITF-ew, or MITF-Wh for 24 hours before being treated with 55 μg/ml CHX. The amount of MITF protein was then compared by western blot using FLAG antibody. Actin was used as a loading control. The band intensities were quantified using ImageJ software. (L) and (M) Non-linear regression (one-phase decay) and half-life analysis of the indicated pS73- and S73-MITF proteins over time after CHX treatment. The relative MITF protein levels to T0 were calculated, and non-linear regression analysis was performed. Error bars represent SEM of at least three independent experiments. Statistically significant differences (Student’s t-test) are indicated by * p< 0.05, *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant.

Figure 2:. The carboxyl-domain of Mitf controls RNA and protein levels as well as its subcellular localization

(A) Western blot analysis of the Mitf-Flag proteins upon cycloheximide treatment. The dox-inducible A375P cells expressing the MITF-WT, MITF-sp, and MITF-sl proteins were treated with doxycycline for 24h to induce similar expression of the indicated mutant MITF proteins before treating them with 40 μg/ml cycloheximide (CHX) for 0, 1, 2, and 3 hours. The blots were stained using Flag antibody and protein quantitated using the odyssey imager and ImageJ. Actin was used as a loading control. (B), (C) Non-linear regression (one-phase decay) and half-life analysis of the indicated pS73- and S73-MITF proteins over time after CHX treatment in A375P melanoma cells. The relative MITF protein levels to T0 were calculated, and non-linear regression analysis was performed. Error bars represent SEM of at least three independent experiments. Statistically significant differences (Student’s t-test) are indicated by *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant. (D) Western blot analysis of subcellular fractions isolated from A375P melanoma cells induced for 24 hours to overexpress different MITF mutant proteins. MITF-WT, MITF-Wh, MITF-sp, and MITF-sl protein in whole cell lysate (W), cytoplasmic (C), and nuclear (N) fractions were visualized using FLAG antibody. Actin and γH2AX were loading controls for cytoplasmic and nuclear fractions, respectively. (E) Intensities of the indicated pS73- and S73-MITF protein bands in the cytoplasmic and nuclear fraction from the western blot analysis in (E) were quantified separately with ImageJ software and are depicted as percentages of the total amount of protein present in the two fractions. Error bars represent SEM of three independent experiments. Statistically significant differences (Student’s t-test) are indicated by * p< 0.05, *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant. (F) Western blot analysis of subcellular fractions isolated from A375P cells transiently co-overexpressing the MITF-sl protein with the MITF-Wh, MITF-mi, and MITF-ew mutant MITF proteins. MITF proteins in cytoplasmic (C) and nuclear (N) fractions were visualized using FLAG antibody. Actin and γH2AX were loading controls for cytoplasmic and nuclear fractions, respectively. The MITF-sl protein migrates as a doublet at 50–55 kDa, whereas the other mutants migrate at 65–70 kDa. (G) The intensities of the pS73- and S73- MITF-sl protein in the cytoplasmic and nuclear fractions from western blot analysis (G) were quantified separately with ImageJ software and are depicted as percentages of the total protein present in the two fractions. Error bars represent SEM of at least three independent experiments. Statistically significant differences (Student’s t-test) are indicated by * p< 0.05, *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant. (H) Quantification of band intensities of the pS73- and S73-versions of the MITF-Wh, MITF-mi, and MITF-ew proteins as determined from western blot analysis (G) in the nuclear fractions of A375P cells transiently co-overexpressing the MITF-sl protein with the indicated MITF mutant proteins. The intensities are depicted as percentages of the total amount of protein present in the two fractions. Error bars represent SEM of at least three independent experiments. Statistically significant differences (Student’s t-test) are indicated by * p< 0.05, *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant. (I), (J), and (K) Western blot analysis of the degradation of the MITF-sl protein in the presence of non-DNA binding MITF mutations (MITF-Wh, MITF-mi, and MITF-ew). The A375P cells were transiently co-transfected with MITF-sl and either MITF-mi, MITF-ew, or MITF-Wh for 24 hours before being treated with 55 μg/ml CHX. The amount of MITF protein was then compared by western blot using FLAG antibody. Actin was used as a loading control. The band intensities were quantified using ImageJ software. (L) and (M) Non-linear regression (one-phase decay) and half-life analysis of the indicated pS73- and S73-MITF proteins over time after CHX treatment. The relative MITF protein levels to T0 were calculated, and non-linear regression analysis was performed. Error bars represent SEM of at least three independent experiments. Statistically significant differences (Student’s t-test) are indicated by * p< 0.05, *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant.

Figure 2:. The carboxyl-domain of Mitf controls RNA and protein levels as well as its subcellular localization

(A) Western blot analysis of the Mitf-Flag proteins upon cycloheximide treatment. The dox-inducible A375P cells expressing the MITF-WT, MITF-sp, and MITF-sl proteins were treated with doxycycline for 24h to induce similar expression of the indicated mutant MITF proteins before treating them with 40 μg/ml cycloheximide (CHX) for 0, 1, 2, and 3 hours. The blots were stained using Flag antibody and protein quantitated using the odyssey imager and ImageJ. Actin was used as a loading control. (B), (C) Non-linear regression (one-phase decay) and half-life analysis of the indicated pS73- and S73-MITF proteins over time after CHX treatment in A375P melanoma cells. The relative MITF protein levels to T0 were calculated, and non-linear regression analysis was performed. Error bars represent SEM of at least three independent experiments. Statistically significant differences (Student’s t-test) are indicated by *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant. (D) Western blot analysis of subcellular fractions isolated from A375P melanoma cells induced for 24 hours to overexpress different MITF mutant proteins. MITF-WT, MITF-Wh, MITF-sp, and MITF-sl protein in whole cell lysate (W), cytoplasmic (C), and nuclear (N) fractions were visualized using FLAG antibody. Actin and γH2AX were loading controls for cytoplasmic and nuclear fractions, respectively. (E) Intensities of the indicated pS73- and S73-MITF protein bands in the cytoplasmic and nuclear fraction from the western blot analysis in (E) were quantified separately with ImageJ software and are depicted as percentages of the total amount of protein present in the two fractions. Error bars represent SEM of three independent experiments. Statistically significant differences (Student’s t-test) are indicated by * p< 0.05, *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant. (F) Western blot analysis of subcellular fractions isolated from A375P cells transiently co-overexpressing the MITF-sl protein with the MITF-Wh, MITF-mi, and MITF-ew mutant MITF proteins. MITF proteins in cytoplasmic (C) and nuclear (N) fractions were visualized using FLAG antibody. Actin and γH2AX were loading controls for cytoplasmic and nuclear fractions, respectively. The MITF-sl protein migrates as a doublet at 50–55 kDa, whereas the other mutants migrate at 65–70 kDa. (G) The intensities of the pS73- and S73- MITF-sl protein in the cytoplasmic and nuclear fractions from western blot analysis (G) were quantified separately with ImageJ software and are depicted as percentages of the total protein present in the two fractions. Error bars represent SEM of at least three independent experiments. Statistically significant differences (Student’s t-test) are indicated by * p< 0.05, *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant. (H) Quantification of band intensities of the pS73- and S73-versions of the MITF-Wh, MITF-mi, and MITF-ew proteins as determined from western blot analysis (G) in the nuclear fractions of A375P cells transiently co-overexpressing the MITF-sl protein with the indicated MITF mutant proteins. The intensities are depicted as percentages of the total amount of protein present in the two fractions. Error bars represent SEM of at least three independent experiments. Statistically significant differences (Student’s t-test) are indicated by * p< 0.05, *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant. (I), (J), and (K) Western blot analysis of the degradation of the MITF-sl protein in the presence of non-DNA binding MITF mutations (MITF-Wh, MITF-mi, and MITF-ew). The A375P cells were transiently co-transfected with MITF-sl and either MITF-mi, MITF-ew, or MITF-Wh for 24 hours before being treated with 55 μg/ml CHX. The amount of MITF protein was then compared by western blot using FLAG antibody. Actin was used as a loading control. The band intensities were quantified using ImageJ software. (L) and (M) Non-linear regression (one-phase decay) and half-life analysis of the indicated pS73- and S73-MITF proteins over time after CHX treatment. The relative MITF protein levels to T0 were calculated, and non-linear regression analysis was performed. Error bars represent SEM of at least three independent experiments. Statistically significant differences (Student’s t-test) are indicated by * p< 0.05, *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant.

Figure 3:. The carboxyl-domains of Mitf control its nuclear localization and stability

(A) Schematic of MITF-sp truncation constructs. C-term truncations were generated by introducing stop codons at position Q326 or L378 or by deleting fragments 326–377 or 316–326. MITF-sp-326* introduces a stop-codon at residue 326 and therefore contains the SUMo-site at 316; MITF-sp-Δ326–377 lacks the tentative activation domain AD3; MITF-sp-Δ316–326 lacks the SUMo-site and adjacent amino acids; MITF-sp-378* lacks the series of phosphorylation sites at the carboxyl-end of the protein. (B) Western blot analysis of subcellular fractions isolated from A375P melanoma cells induced to overexpress the different MITF mutant proteins fusioned with Flag-tag at C terminus for 24 hours. MITF-WT, MITF-sl, MITF-sp-326*, MITFmi-sp-378*, and MITF-sp-Δ326–377 in cytoplasmic (C) and nuclear (N) fractions were visualized using FLAG antibody. Actin and γH2AX were loading controls for cytoplasmic and nuclear fractions, respectively. (C) The intensities of the indicated pS73 MITF and S73 MITF proteins from the cytoplasmic and nuclear fractions of the western blot analysis in (B) were quantified separately with ImageJ software and are depicted as percentages of the total amount of protein present in the two fractions. Error bars represent SEM of three independent experiments. Statistically significant differences (Student’s t-test) are indicated by * p< 0.05, *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant. (D) Western blot analysis of subcellular fractions isolated from A375P melanoma cells induced for 24 hours to overexpress the different MITF mutant proteins before treatment with TPA at 200nM for 1 hour. MITF-WT, MITF-sl, MITF-sp-326*, MITF-sp-Δ326–377, MITF-sp-Δ316–326, and MITF-sp-378* protein in cytoplasmic (C) and nuclear (N) fractions were visualized using FLAG antibody. Actin or GAPDH and γH2AX or H3K27me3 were loading controls for cytoplasmic and nuclear fractions, respectively. (E) Intensities of the indicated pS73-MITF proteins from the western blot analysis in (D) in the cytoplasmic and nuclear fractions from the cell treated with TPA were quantified separately with ImageJ software and are depicted as percentages of the total amount of protein present in the two fractions. Error bars represent SEM of three independent experiments. Statistically significant differences (Student’s t-test) are indicated by * p< 0.05, *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant. (F) Western blot analysis of the MITF proteins from dox-induced A375P cells after treating them with 40 μg/ml CHX for 0, 1, 2, and 3 hours. The MITF proteins were visualized by western blot using FLAG antibody. Actin was used as a loading control. The band intensities were quantified using ImageJ software. (G) and (H) Non-linear regression (one-phase decay) and half-life analysis of the indicated pS73- and S73-MITF proteins over time after CHX treatment. The MITF protein levels relative to T0 were calculated, and non-linear regression analysis was performed. Error bars represent SEM of at least three independent experiments. Statistically significant differences (Student’s t-test) are indicated by * p< 0.05, *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant.

Figure 3:. The carboxyl-domains of Mitf control its nuclear localization and stability

(A) Schematic of MITF-sp truncation constructs. C-term truncations were generated by introducing stop codons at position Q326 or L378 or by deleting fragments 326–377 or 316–326. MITF-sp-326* introduces a stop-codon at residue 326 and therefore contains the SUMo-site at 316; MITF-sp-Δ326–377 lacks the tentative activation domain AD3; MITF-sp-Δ316–326 lacks the SUMo-site and adjacent amino acids; MITF-sp-378* lacks the series of phosphorylation sites at the carboxyl-end of the protein. (B) Western blot analysis of subcellular fractions isolated from A375P melanoma cells induced to overexpress the different MITF mutant proteins fusioned with Flag-tag at C terminus for 24 hours. MITF-WT, MITF-sl, MITF-sp-326*, MITFmi-sp-378*, and MITF-sp-Δ326–377 in cytoplasmic (C) and nuclear (N) fractions were visualized using FLAG antibody. Actin and γH2AX were loading controls for cytoplasmic and nuclear fractions, respectively. (C) The intensities of the indicated pS73 MITF and S73 MITF proteins from the cytoplasmic and nuclear fractions of the western blot analysis in (B) were quantified separately with ImageJ software and are depicted as percentages of the total amount of protein present in the two fractions. Error bars represent SEM of three independent experiments. Statistically significant differences (Student’s t-test) are indicated by * p< 0.05, *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant. (D) Western blot analysis of subcellular fractions isolated from A375P melanoma cells induced for 24 hours to overexpress the different MITF mutant proteins before treatment with TPA at 200nM for 1 hour. MITF-WT, MITF-sl, MITF-sp-326*, MITF-sp-Δ326–377, MITF-sp-Δ316–326, and MITF-sp-378* protein in cytoplasmic (C) and nuclear (N) fractions were visualized using FLAG antibody. Actin or GAPDH and γH2AX or H3K27me3 were loading controls for cytoplasmic and nuclear fractions, respectively. (E) Intensities of the indicated pS73-MITF proteins from the western blot analysis in (D) in the cytoplasmic and nuclear fractions from the cell treated with TPA were quantified separately with ImageJ software and are depicted as percentages of the total amount of protein present in the two fractions. Error bars represent SEM of three independent experiments. Statistically significant differences (Student’s t-test) are indicated by * p< 0.05, *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant. (F) Western blot analysis of the MITF proteins from dox-induced A375P cells after treating them with 40 μg/ml CHX for 0, 1, 2, and 3 hours. The MITF proteins were visualized by western blot using FLAG antibody. Actin was used as a loading control. The band intensities were quantified using ImageJ software. (G) and (H) Non-linear regression (one-phase decay) and half-life analysis of the indicated pS73- and S73-MITF proteins over time after CHX treatment. The MITF protein levels relative to T0 were calculated, and non-linear regression analysis was performed. Error bars represent SEM of at least three independent experiments. Statistically significant differences (Student’s t-test) are indicated by * p< 0.05, *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant.

Figure 3:. The carboxyl-domains of Mitf control its nuclear localization and stability

(A) Schematic of MITF-sp truncation constructs. C-term truncations were generated by introducing stop codons at position Q326 or L378 or by deleting fragments 326–377 or 316–326. MITF-sp-326* introduces a stop-codon at residue 326 and therefore contains the SUMo-site at 316; MITF-sp-Δ326–377 lacks the tentative activation domain AD3; MITF-sp-Δ316–326 lacks the SUMo-site and adjacent amino acids; MITF-sp-378* lacks the series of phosphorylation sites at the carboxyl-end of the protein. (B) Western blot analysis of subcellular fractions isolated from A375P melanoma cells induced to overexpress the different MITF mutant proteins fusioned with Flag-tag at C terminus for 24 hours. MITF-WT, MITF-sl, MITF-sp-326*, MITFmi-sp-378*, and MITF-sp-Δ326–377 in cytoplasmic (C) and nuclear (N) fractions were visualized using FLAG antibody. Actin and γH2AX were loading controls for cytoplasmic and nuclear fractions, respectively. (C) The intensities of the indicated pS73 MITF and S73 MITF proteins from the cytoplasmic and nuclear fractions of the western blot analysis in (B) were quantified separately with ImageJ software and are depicted as percentages of the total amount of protein present in the two fractions. Error bars represent SEM of three independent experiments. Statistically significant differences (Student’s t-test) are indicated by * p< 0.05, *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant. (D) Western blot analysis of subcellular fractions isolated from A375P melanoma cells induced for 24 hours to overexpress the different MITF mutant proteins before treatment with TPA at 200nM for 1 hour. MITF-WT, MITF-sl, MITF-sp-326*, MITF-sp-Δ326–377, MITF-sp-Δ316–326, and MITF-sp-378* protein in cytoplasmic (C) and nuclear (N) fractions were visualized using FLAG antibody. Actin or GAPDH and γH2AX or H3K27me3 were loading controls for cytoplasmic and nuclear fractions, respectively. (E) Intensities of the indicated pS73-MITF proteins from the western blot analysis in (D) in the cytoplasmic and nuclear fractions from the cell treated with TPA were quantified separately with ImageJ software and are depicted as percentages of the total amount of protein present in the two fractions. Error bars represent SEM of three independent experiments. Statistically significant differences (Student’s t-test) are indicated by * p< 0.05, *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant. (F) Western blot analysis of the MITF proteins from dox-induced A375P cells after treating them with 40 μg/ml CHX for 0, 1, 2, and 3 hours. The MITF proteins were visualized by western blot using FLAG antibody. Actin was used as a loading control. The band intensities were quantified using ImageJ software. (G) and (H) Non-linear regression (one-phase decay) and half-life analysis of the indicated pS73- and S73-MITF proteins over time after CHX treatment. The MITF protein levels relative to T0 were calculated, and non-linear regression analysis was performed. Error bars represent SEM of at least three independent experiments. Statistically significant differences (Student’s t-test) are indicated by * p< 0.05, *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant.

Figure 3:. The carboxyl-domains of Mitf control its nuclear localization and stability

(A) Schematic of MITF-sp truncation constructs. C-term truncations were generated by introducing stop codons at position Q326 or L378 or by deleting fragments 326–377 or 316–326. MITF-sp-326* introduces a stop-codon at residue 326 and therefore contains the SUMo-site at 316; MITF-sp-Δ326–377 lacks the tentative activation domain AD3; MITF-sp-Δ316–326 lacks the SUMo-site and adjacent amino acids; MITF-sp-378* lacks the series of phosphorylation sites at the carboxyl-end of the protein. (B) Western blot analysis of subcellular fractions isolated from A375P melanoma cells induced to overexpress the different MITF mutant proteins fusioned with Flag-tag at C terminus for 24 hours. MITF-WT, MITF-sl, MITF-sp-326*, MITFmi-sp-378*, and MITF-sp-Δ326–377 in cytoplasmic (C) and nuclear (N) fractions were visualized using FLAG antibody. Actin and γH2AX were loading controls for cytoplasmic and nuclear fractions, respectively. (C) The intensities of the indicated pS73 MITF and S73 MITF proteins from the cytoplasmic and nuclear fractions of the western blot analysis in (B) were quantified separately with ImageJ software and are depicted as percentages of the total amount of protein present in the two fractions. Error bars represent SEM of three independent experiments. Statistically significant differences (Student’s t-test) are indicated by * p< 0.05, *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant. (D) Western blot analysis of subcellular fractions isolated from A375P melanoma cells induced for 24 hours to overexpress the different MITF mutant proteins before treatment with TPA at 200nM for 1 hour. MITF-WT, MITF-sl, MITF-sp-326*, MITF-sp-Δ326–377, MITF-sp-Δ316–326, and MITF-sp-378* protein in cytoplasmic (C) and nuclear (N) fractions were visualized using FLAG antibody. Actin or GAPDH and γH2AX or H3K27me3 were loading controls for cytoplasmic and nuclear fractions, respectively. (E) Intensities of the indicated pS73-MITF proteins from the western blot analysis in (D) in the cytoplasmic and nuclear fractions from the cell treated with TPA were quantified separately with ImageJ software and are depicted as percentages of the total amount of protein present in the two fractions. Error bars represent SEM of three independent experiments. Statistically significant differences (Student’s t-test) are indicated by * p< 0.05, *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant. (F) Western blot analysis of the MITF proteins from dox-induced A375P cells after treating them with 40 μg/ml CHX for 0, 1, 2, and 3 hours. The MITF proteins were visualized by western blot using FLAG antibody. Actin was used as a loading control. The band intensities were quantified using ImageJ software. (G) and (H) Non-linear regression (one-phase decay) and half-life analysis of the indicated pS73- and S73-MITF proteins over time after CHX treatment. The MITF protein levels relative to T0 were calculated, and non-linear regression analysis was performed. Error bars represent SEM of at least three independent experiments. Statistically significant differences (Student’s t-test) are indicated by * p< 0.05, *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant.

Figure 4:. MITF is mainly degraded through the proteasome pathway in the nucleus.

(A) Western blot analysis of the MITF-WT, MITF-sp, and MITF-sl proteins. Expression was induced for 24 hours in A375P cells treated with 50 μg/ml CHX in the presence of either DMSo or 40 μg/ml MG132 or 0.1 μg/ml Baf-A1 for 3 hours. The MITF protein was then visualized by western blot using FLAG antibody. Actin was used as a loading control. The band intensities were quantified using ImageJ software. (C) Western blot analysis of the MITF-WT, MITF-sp, and MITF-sl proteins. Expression was induced for 24 hours in A375P cells treated with either DMSo or 40 μg/ml MG132 or 0.1 μg/ml Baf-A1 for 3 hours. The MITF protein was then visualized by western blot using FLAG antibody. Actin was used as a loading control. The band intensities were quantified using ImageJ software. (B) and (D) The indicated pS73- and S73-MITF protein band intensities from western blot analysis (A) and (C), respectively, were quantified separately with ImageJ software and are depicted relative to DMSo. Error bars represent SEM of at least three independent experiments. Statistically significant differences (Student’s t-test) are indicated by * p< 0.05, *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant. (E) Western blot analysis of subcellular fractions isolated from A375P melanoma cells induced to overexpress MITF-WT protein before treating with either 200nM TPA for 1 or 4 hours or 40 μg/ml MG132 for 3 hours or 200nM TPA for 1 hour and then adding 40 μg/ml MG132 for the next 3 hours. MITF-WT protein in cytoplasmic (C) and nuclear (N) fractions were visualized using FLAG antibody. GAPDH and γH2AX were loading controls for cytoplasmic and nuclear fractions, respectively. (F) Western blot analysis of the stability of the MITF-WT and MITF-sl mutant proteins after knocking down AKIRIN2, a key regulator of the nuclear import of proteasomes, for 24 hours and then inducing MITF expression using dox for 6 hours. The inducible A375P cells were treated with 40 μg/ml CHX for 0, 1, 2, and 3 hours. The MITF proteins were then visualized by western blot using FLAG antibody. Actin was used as a loading control. The band intensities were quantified using ImageJ software. (G) The intensities of the indicated pS73- and S73-MITF protein bands were quantified from western blot analysis in (F) with ImageJ software and are depicted as relative protein expression to DMSo. Error bars represent SEM of three independent experiments. Statistically significant differences (Student’s t-test) are indicated by * p< 0.05, *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant.

Figure 4:. MITF is mainly degraded through the proteasome pathway in the nucleus.

(A) Western blot analysis of the MITF-WT, MITF-sp, and MITF-sl proteins. Expression was induced for 24 hours in A375P cells treated with 50 μg/ml CHX in the presence of either DMSo or 40 μg/ml MG132 or 0.1 μg/ml Baf-A1 for 3 hours. The MITF protein was then visualized by western blot using FLAG antibody. Actin was used as a loading control. The band intensities were quantified using ImageJ software. (C) Western blot analysis of the MITF-WT, MITF-sp, and MITF-sl proteins. Expression was induced for 24 hours in A375P cells treated with either DMSo or 40 μg/ml MG132 or 0.1 μg/ml Baf-A1 for 3 hours. The MITF protein was then visualized by western blot using FLAG antibody. Actin was used as a loading control. The band intensities were quantified using ImageJ software. (B) and (D) The indicated pS73- and S73-MITF protein band intensities from western blot analysis (A) and (C), respectively, were quantified separately with ImageJ software and are depicted relative to DMSo. Error bars represent SEM of at least three independent experiments. Statistically significant differences (Student’s t-test) are indicated by * p< 0.05, *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant. (E) Western blot analysis of subcellular fractions isolated from A375P melanoma cells induced to overexpress MITF-WT protein before treating with either 200nM TPA for 1 or 4 hours or 40 μg/ml MG132 for 3 hours or 200nM TPA for 1 hour and then adding 40 μg/ml MG132 for the next 3 hours. MITF-WT protein in cytoplasmic (C) and nuclear (N) fractions were visualized using FLAG antibody. GAPDH and γH2AX were loading controls for cytoplasmic and nuclear fractions, respectively. (F) Western blot analysis of the stability of the MITF-WT and MITF-sl mutant proteins after knocking down AKIRIN2, a key regulator of the nuclear import of proteasomes, for 24 hours and then inducing MITF expression using dox for 6 hours. The inducible A375P cells were treated with 40 μg/ml CHX for 0, 1, 2, and 3 hours. The MITF proteins were then visualized by western blot using FLAG antibody. Actin was used as a loading control. The band intensities were quantified using ImageJ software. (G) The intensities of the indicated pS73- and S73-MITF protein bands were quantified from western blot analysis in (F) with ImageJ software and are depicted as relative protein expression to DMSo. Error bars represent SEM of three independent experiments. Statistically significant differences (Student’s t-test) are indicated by * p< 0.05, *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant.

Figure 4:. MITF is mainly degraded through the proteasome pathway in the nucleus.

(A) Western blot analysis of the MITF-WT, MITF-sp, and MITF-sl proteins. Expression was induced for 24 hours in A375P cells treated with 50 μg/ml CHX in the presence of either DMSo or 40 μg/ml MG132 or 0.1 μg/ml Baf-A1 for 3 hours. The MITF protein was then visualized by western blot using FLAG antibody. Actin was used as a loading control. The band intensities were quantified using ImageJ software. (C) Western blot analysis of the MITF-WT, MITF-sp, and MITF-sl proteins. Expression was induced for 24 hours in A375P cells treated with either DMSo or 40 μg/ml MG132 or 0.1 μg/ml Baf-A1 for 3 hours. The MITF protein was then visualized by western blot using FLAG antibody. Actin was used as a loading control. The band intensities were quantified using ImageJ software. (B) and (D) The indicated pS73- and S73-MITF protein band intensities from western blot analysis (A) and (C), respectively, were quantified separately with ImageJ software and are depicted relative to DMSo. Error bars represent SEM of at least three independent experiments. Statistically significant differences (Student’s t-test) are indicated by * p< 0.05, *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant. (E) Western blot analysis of subcellular fractions isolated from A375P melanoma cells induced to overexpress MITF-WT protein before treating with either 200nM TPA for 1 or 4 hours or 40 μg/ml MG132 for 3 hours or 200nM TPA for 1 hour and then adding 40 μg/ml MG132 for the next 3 hours. MITF-WT protein in cytoplasmic (C) and nuclear (N) fractions were visualized using FLAG antibody. GAPDH and γH2AX were loading controls for cytoplasmic and nuclear fractions, respectively. (F) Western blot analysis of the stability of the MITF-WT and MITF-sl mutant proteins after knocking down AKIRIN2, a key regulator of the nuclear import of proteasomes, for 24 hours and then inducing MITF expression using dox for 6 hours. The inducible A375P cells were treated with 40 μg/ml CHX for 0, 1, 2, and 3 hours. The MITF proteins were then visualized by western blot using FLAG antibody. Actin was used as a loading control. The band intensities were quantified using ImageJ software. (G) The intensities of the indicated pS73- and S73-MITF protein bands were quantified from western blot analysis in (F) with ImageJ software and are depicted as relative protein expression to DMSo. Error bars represent SEM of three independent experiments. Statistically significant differences (Student’s t-test) are indicated by * p< 0.05, *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant.

Figure 4:. MITF is mainly degraded through the proteasome pathway in the nucleus.

(A) Western blot analysis of the MITF-WT, MITF-sp, and MITF-sl proteins. Expression was induced for 24 hours in A375P cells treated with 50 μg/ml CHX in the presence of either DMSo or 40 μg/ml MG132 or 0.1 μg/ml Baf-A1 for 3 hours. The MITF protein was then visualized by western blot using FLAG antibody. Actin was used as a loading control. The band intensities were quantified using ImageJ software. (C) Western blot analysis of the MITF-WT, MITF-sp, and MITF-sl proteins. Expression was induced for 24 hours in A375P cells treated with either DMSo or 40 μg/ml MG132 or 0.1 μg/ml Baf-A1 for 3 hours. The MITF protein was then visualized by western blot using FLAG antibody. Actin was used as a loading control. The band intensities were quantified using ImageJ software. (B) and (D) The indicated pS73- and S73-MITF protein band intensities from western blot analysis (A) and (C), respectively, were quantified separately with ImageJ software and are depicted relative to DMSo. Error bars represent SEM of at least three independent experiments. Statistically significant differences (Student’s t-test) are indicated by * p< 0.05, *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant. (E) Western blot analysis of subcellular fractions isolated from A375P melanoma cells induced to overexpress MITF-WT protein before treating with either 200nM TPA for 1 or 4 hours or 40 μg/ml MG132 for 3 hours or 200nM TPA for 1 hour and then adding 40 μg/ml MG132 for the next 3 hours. MITF-WT protein in cytoplasmic (C) and nuclear (N) fractions were visualized using FLAG antibody. GAPDH and γH2AX were loading controls for cytoplasmic and nuclear fractions, respectively. (F) Western blot analysis of the stability of the MITF-WT and MITF-sl mutant proteins after knocking down AKIRIN2, a key regulator of the nuclear import of proteasomes, for 24 hours and then inducing MITF expression using dox for 6 hours. The inducible A375P cells were treated with 40 μg/ml CHX for 0, 1, 2, and 3 hours. The MITF proteins were then visualized by western blot using FLAG antibody. Actin was used as a loading control. The band intensities were quantified using ImageJ software. (G) The intensities of the indicated pS73- and S73-MITF protein bands were quantified from western blot analysis in (F) with ImageJ software and are depicted as relative protein expression to DMSo. Error bars represent SEM of three independent experiments. Statistically significant differences (Student’s t-test) are indicated by * p< 0.05, *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant.

Figure 5:. The interplay between SUMoylation at K316 and phosphorylation site at S409 in regulating MITF protein stability and localization.

(A) Western blot analysis of subcellular fractions isolated from A375P melanoma cells induced for 24 hours to overexpress the indicated MITF mutant proteins. The MITF proteins in cytoplasmic (C) and nuclear (N) fractions were visualized using FLAG antibody. Actin or GAPDH and H3K27me3 were loading controls for cytoplasmic and nuclear fractions, respectively. (B) and (C) The intensities of the indicated pS73- and S73-MITF proteins in the cytoplasmic and nuclear fractions from western blot analysis in (A) were quantified separately with ImageJ software and are depicted as percentages of the total amount of protein present in the two fractions. Error bars represent SEM of three independent experiments. Statistically significant differences (Student’s t-test) are indicated by * p< 0.05, *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant. (D) Western blot analysis of subcellular fractions isolated from A375P melanoma cells induced for 24 hours to overexpress the indicated MITF mutant proteins before treatment with 200nM TPA for 1 hour. The mutant MITF proteins in cytoplasmic (C) and nuclear (N) fractions were visualized using FLAG antibody. GAPDH and H3K27me3 were loading controls for cytoplasmic and nuclear fractions, respectively. (E) The intensities of the indicated pS73-MITF proteins bands in the cytoplasmic and nuclear fractions of the western blot analysis in (D) and (F), respectively, were quantified separately with ImageJ software and are depicted as percentages of the total amount of protein present in the two fractions. Error bars represent SEM of three independent experiments. Statistically significant differences (Student’s t-test) are indicated by * p< 0.05, *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant. (F) and (H) Western blot analysis of the stability of the MITF proteins. The inducible A375P cells were treated with doxycycline for 24h to express the indicated mutant MITF proteins before treating them with 40 μg/ml CHX for 0, 1, 2, and 3 hours. The MITF protein was then compared by western blot using FLAG antibody. Actin was used as a loading control. The band intensities were quantified using ImageJ software. (G) and (I) Half-life analysis of the pS73- and S73-MITF proteins over time after CHX treatment. The MITF protein levels relative to T0 were calculated, and non-linear regression analysis was performed. Error bars represent SEM of at least three independent experiments. Statistically significant differences (Student’s t-test) are indicated by * p< 0.05, *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant.

Figure 5:. The interplay between SUMoylation at K316 and phosphorylation site at S409 in regulating MITF protein stability and localization.

(A) Western blot analysis of subcellular fractions isolated from A375P melanoma cells induced for 24 hours to overexpress the indicated MITF mutant proteins. The MITF proteins in cytoplasmic (C) and nuclear (N) fractions were visualized using FLAG antibody. Actin or GAPDH and H3K27me3 were loading controls for cytoplasmic and nuclear fractions, respectively. (B) and (C) The intensities of the indicated pS73- and S73-MITF proteins in the cytoplasmic and nuclear fractions from western blot analysis in (A) were quantified separately with ImageJ software and are depicted as percentages of the total amount of protein present in the two fractions. Error bars represent SEM of three independent experiments. Statistically significant differences (Student’s t-test) are indicated by * p< 0.05, *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant. (D) Western blot analysis of subcellular fractions isolated from A375P melanoma cells induced for 24 hours to overexpress the indicated MITF mutant proteins before treatment with 200nM TPA for 1 hour. The mutant MITF proteins in cytoplasmic (C) and nuclear (N) fractions were visualized using FLAG antibody. GAPDH and H3K27me3 were loading controls for cytoplasmic and nuclear fractions, respectively. (E) The intensities of the indicated pS73-MITF proteins bands in the cytoplasmic and nuclear fractions of the western blot analysis in (D) and (F), respectively, were quantified separately with ImageJ software and are depicted as percentages of the total amount of protein present in the two fractions. Error bars represent SEM of three independent experiments. Statistically significant differences (Student’s t-test) are indicated by * p< 0.05, *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant. (F) and (H) Western blot analysis of the stability of the MITF proteins. The inducible A375P cells were treated with doxycycline for 24h to express the indicated mutant MITF proteins before treating them with 40 μg/ml CHX for 0, 1, 2, and 3 hours. The MITF protein was then compared by western blot using FLAG antibody. Actin was used as a loading control. The band intensities were quantified using ImageJ software. (G) and (I) Half-life analysis of the pS73- and S73-MITF proteins over time after CHX treatment. The MITF protein levels relative to T0 were calculated, and non-linear regression analysis was performed. Error bars represent SEM of at least three independent experiments. Statistically significant differences (Student’s t-test) are indicated by * p< 0.05, *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant.

Figure 5:. The interplay between SUMoylation at K316 and phosphorylation site at S409 in regulating MITF protein stability and localization.

(A) Western blot analysis of subcellular fractions isolated from A375P melanoma cells induced for 24 hours to overexpress the indicated MITF mutant proteins. The MITF proteins in cytoplasmic (C) and nuclear (N) fractions were visualized using FLAG antibody. Actin or GAPDH and H3K27me3 were loading controls for cytoplasmic and nuclear fractions, respectively. (B) and (C) The intensities of the indicated pS73- and S73-MITF proteins in the cytoplasmic and nuclear fractions from western blot analysis in (A) were quantified separately with ImageJ software and are depicted as percentages of the total amount of protein present in the two fractions. Error bars represent SEM of three independent experiments. Statistically significant differences (Student’s t-test) are indicated by * p< 0.05, *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant. (D) Western blot analysis of subcellular fractions isolated from A375P melanoma cells induced for 24 hours to overexpress the indicated MITF mutant proteins before treatment with 200nM TPA for 1 hour. The mutant MITF proteins in cytoplasmic (C) and nuclear (N) fractions were visualized using FLAG antibody. GAPDH and H3K27me3 were loading controls for cytoplasmic and nuclear fractions, respectively. (E) The intensities of the indicated pS73-MITF proteins bands in the cytoplasmic and nuclear fractions of the western blot analysis in (D) and (F), respectively, were quantified separately with ImageJ software and are depicted as percentages of the total amount of protein present in the two fractions. Error bars represent SEM of three independent experiments. Statistically significant differences (Student’s t-test) are indicated by * p< 0.05, *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant. (F) and (H) Western blot analysis of the stability of the MITF proteins. The inducible A375P cells were treated with doxycycline for 24h to express the indicated mutant MITF proteins before treating them with 40 μg/ml CHX for 0, 1, 2, and 3 hours. The MITF protein was then compared by western blot using FLAG antibody. Actin was used as a loading control. The band intensities were quantified using ImageJ software. (G) and (I) Half-life analysis of the pS73- and S73-MITF proteins over time after CHX treatment. The MITF protein levels relative to T0 were calculated, and non-linear regression analysis was performed. Error bars represent SEM of at least three independent experiments. Statistically significant differences (Student’s t-test) are indicated by * p< 0.05, *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant.

Figure 5:. The interplay between SUMoylation at K316 and phosphorylation site at S409 in regulating MITF protein stability and localization.

(A) Western blot analysis of subcellular fractions isolated from A375P melanoma cells induced for 24 hours to overexpress the indicated MITF mutant proteins. The MITF proteins in cytoplasmic (C) and nuclear (N) fractions were visualized using FLAG antibody. Actin or GAPDH and H3K27me3 were loading controls for cytoplasmic and nuclear fractions, respectively. (B) and (C) The intensities of the indicated pS73- and S73-MITF proteins in the cytoplasmic and nuclear fractions from western blot analysis in (A) were quantified separately with ImageJ software and are depicted as percentages of the total amount of protein present in the two fractions. Error bars represent SEM of three independent experiments. Statistically significant differences (Student’s t-test) are indicated by * p< 0.05, *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant. (D) Western blot analysis of subcellular fractions isolated from A375P melanoma cells induced for 24 hours to overexpress the indicated MITF mutant proteins before treatment with 200nM TPA for 1 hour. The mutant MITF proteins in cytoplasmic (C) and nuclear (N) fractions were visualized using FLAG antibody. GAPDH and H3K27me3 were loading controls for cytoplasmic and nuclear fractions, respectively. (E) The intensities of the indicated pS73-MITF proteins bands in the cytoplasmic and nuclear fractions of the western blot analysis in (D) and (F), respectively, were quantified separately with ImageJ software and are depicted as percentages of the total amount of protein present in the two fractions. Error bars represent SEM of three independent experiments. Statistically significant differences (Student’s t-test) are indicated by * p< 0.05, *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant. (F) and (H) Western blot analysis of the stability of the MITF proteins. The inducible A375P cells were treated with doxycycline for 24h to express the indicated mutant MITF proteins before treating them with 40 μg/ml CHX for 0, 1, 2, and 3 hours. The MITF protein was then compared by western blot using FLAG antibody. Actin was used as a loading control. The band intensities were quantified using ImageJ software. (G) and (I) Half-life analysis of the pS73- and S73-MITF proteins over time after CHX treatment. The MITF protein levels relative to T0 were calculated, and non-linear regression analysis was performed. Error bars represent SEM of at least three independent experiments. Statistically significant differences (Student’s t-test) are indicated by * p< 0.05, *p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00, and ns not significant.

Figure 6:. Inter- and intramolecular FRET indicates MITF C-end IDRs are proximal.

(A) and (B) Single-molecule Förster resonance energy transfer histograms of dimeric MITF fluorescently labeled at residues C306 and C419. Intermolecular FRET (A), fitted Gaussian population mean, E=0.66, and intramolecular FRET (B), fitted Gaussian population mean, E=0.3. (C) Single-molecule Förster resonance energy transfer histograms of MITF single-labeled pairs, labeled with acceptor or donor as indicated. FRET population fitted with a Gaussian distribution in blue with donor-only events in grey. (D) Fluorescence lifetime analysis of inter- and intramolecular distances of the C-terminal IDR of MITF. The 2-D plot shows the lifetime of the donor in the presence of the acceptor (${\tau }_D^{\mathit{\text{FRET}}}$), relative to the donor fluorescence in its absence (${\tau }_D^{\mathit{\text{Donly}}}$), plotted against the FRET transfer efficiency for each burst. The solid black line shows the expected relationship for a static distance, while the grey line shows the expected relationship for a dynamic chain with a Gaussian distribution of distances.

Figure 6:. Inter- and intramolecular FRET indicates MITF C-end IDRs are proximal.

(A) and (B) Single-molecule Förster resonance energy transfer histograms of dimeric MITF fluorescently labeled at residues C306 and C419. Intermolecular FRET (A), fitted Gaussian population mean, E=0.66, and intramolecular FRET (B), fitted Gaussian population mean, E=0.3. (C) Single-molecule Förster resonance energy transfer histograms of MITF single-labeled pairs, labeled with acceptor or donor as indicated. FRET population fitted with a Gaussian distribution in blue with donor-only events in grey. (D) Fluorescence lifetime analysis of inter- and intramolecular distances of the C-terminal IDR of MITF. The 2-D plot shows the lifetime of the donor in the presence of the acceptor (${\tau }_D^{\mathit{\text{FRET}}}$), relative to the donor fluorescence in its absence (${\tau }_D^{\mathit{\text{Donly}}}$), plotted against the FRET transfer efficiency for each burst. The solid black line shows the expected relationship for a static distance, while the grey line shows the expected relationship for a dynamic chain with a Gaussian distribution of distances.

Figure 6:. Inter- and intramolecular FRET indicates MITF C-end IDRs are proximal.

(A) and (B) Single-molecule Förster resonance energy transfer histograms of dimeric MITF fluorescently labeled at residues C306 and C419. Intermolecular FRET (A), fitted Gaussian population mean, E=0.66, and intramolecular FRET (B), fitted Gaussian population mean, E=0.3. (C) Single-molecule Förster resonance energy transfer histograms of MITF single-labeled pairs, labeled with acceptor or donor as indicated. FRET population fitted with a Gaussian distribution in blue with donor-only events in grey. (D) Fluorescence lifetime analysis of inter- and intramolecular distances of the C-terminal IDR of MITF. The 2-D plot shows the lifetime of the donor in the presence of the acceptor (${\tau }_D^{\mathit{\text{FRET}}}$), relative to the donor fluorescence in its absence (${\tau }_D^{\mathit{\text{Donly}}}$), plotted against the FRET transfer efficiency for each burst. The solid black line shows the expected relationship for a static distance, while the grey line shows the expected relationship for a dynamic chain with a Gaussian distribution of distances.