K-Ras Lys-42 is crucial for its signaling, cell migration, and invasion

Abstract

Ras proteins participate in multiple signal cascades, regulating crucial cellular processes, including cell survival, proliferation, and differentiation. We have previously reported that Ras proteins are modified by sumoylation and that Lys-42 plays an important role in mediating the modification. In the current study, we further investigated the role of Lys-42 in regulating cellular activities of K-Ras. Inducible expression of K-Ras^V12 led to the activation of downstream components, including c-RAF, MEK1, and extracellular signal-regulated kinases (ERKs), whereas expression of K-Ras^V12/R42 mutant compromised the activation of the RAF/MEK/ERK signaling axis. Expression of K-Ras^V12/R42 also led to reduced phosphorylation of several other protein kinases, including c-Jun N-terminal kinase (JNK), Chk2, and focal adhesion kinase (FAK). Significantly, K-Ras^V12/R42 expression inhibited cellular migration and invasion in vitro in multiple cell lines, including transformed pancreatic cells. Given that K-Ras plays a crucial role in mediating oncogenesis in the pancreas, we treated transformed pancreatic cells of both BxPC-3 and MiaPaCa-2 with 2-D08, a small ubiquitin-like modifier (SUMO) E2 inhibitor. Treatment with the compound inhibited cell migration in a concentration-dependent manner, which was correlated with a reduced level of K-Ras sumoylation. Moreover, 2-D08 suppressed expression of ZEB1 (a mesenchymal cell marker) with concomitant induction of ZO-1 (an epithelial cell marker). Combined, our studies strongly suggest that posttranslational modification(s), including sumoylation mediated by Lys-42, plays a crucial role in K-Ras activities in vivo.

Keywords: K-Ras; Ras protein; cell invasion; cell migration; domain structure; lysine residue; posttranslational modification (PTM); signaling; sumoylation; transformation.

Figure 1.

Lys-42 is crucial for K-Ras downstream signaling. A, schematic representation of major domains of K-Ras protein. Lys-42 is located between switch I (amino acids 32–38) and II (amino acids 59–67) domains that mediate the interaction with its regulators and effectors. The hypervariable (HVR) domain between amino acids 165 and 188 in the C terminus specifies the membrane localization through extensive posttranslational modifications. B, Tet293/K-Ras^V12 or Tet293/K-Ras^V12/R42 cells were cultured in the presence or absence of Dox. At various times of culture, cells were collected and lysed. Equal amounts of cell lysates were blotted with antibodies to FLAG (K-Ras), pan-Ras, phospho-cRaf338 (p-cRaf388), phospho-MEK (p-MEK), pan-MEK, phospho-ERK (p-ERK), pan-ERK, phospho-AKT (p-AKT), pan-AKT, and β-actin. C, HEK293T cells were transfected with plasmids expressing K-Ras^V12, K-^RasV12/R42, or vector (ev) alone. These cells were also co-transfected with or without SENP1 for 24 h, after which cells were lysed and equal amounts of cell lysates were immunoprecipitated with the anti-FLAG antibody. FLAG immunoprecipitates (IP), along with cell lysate inputs, were blotted for c-Raf, β-actin, and FLAG. In all relevant panels, molecular weight markers are also indicated.

Figure 2.

Lys-42 is important for activation of protein kinases mediating various biological responses. A, selected dot blots of protein kinase array using paired cellular lysates as shown. Representative protein kinases (except for AKT and GSK3) exhibiting significant differences between K-Ras^V12 and K-Ras^V12/R42 are shown. EV, empty vector; CT, control for the array analysis. See Fig. S1 for additional information and details. B, dot blot signals as shown in A (and Fig. S3) were quantified and were then normalized to signals in cells transfected with empty vector. Relative signal intensity is represented by plus signs.

Figure 3.

Lys-42 is essential for mediating cell migration. A, NIH3T3 cells transfected with FLAG-K-Ras^V12, FLAG-K-Ras^V12/R42 expression plasmid, or vector alone for 24 h were subjected to conventional wound-healing assays. Representative images of cells at 0 and 18 h postscratching are shown. B, percentage of wound closure as described in A was quantified. Data are summarized from three independent experiments. Ve, vector-transfected control. C, equal amounts of lysates from cells transfected with various expression plasmids as described in B were blotted with antibodies to FLAG and β-actin, respectively. D, MCF cells were transfected with a plasmid construct expressing FLAG-K-Ras^V12 or FLAG-K-Ras^V12/R42 for 24 h. Empty plasmid vector was also used for transfection as control. Images at 18 h postscratching for various transfections were taken. Representative images are shown. E, percentage of wound closure as described in D was quantified. Data are summarized from three independent experiments. Ve, vector-transfected control. F, MCF7 cells were transfected with a plasmid construct expressing K-Ras^V12 or K-Ras^V12/R42 for 24 h. Empty plasmid vector was also used for transfection as control. Equal amounts of cell lysates were blotted for phospho-cRaf388 (p-cRaf388), phospho-MEK (p-MEK), MEK, phospho-ERK (p-ERK), ERK, Snail, claudin-1, FLAG, and β-actin. In all relevant panels, molecular weight markers are also indicated. Error bars, S.D. *, p < 0.05.

Figure 4.

Lys-42 is essential for mediating migration of transformed pancreatic cells. A, MiaPaCa-2 cells were transfected with various K-Ras expression plasmids as shown for 24 h. GFP expression plasmid was used for co-transfection as control. Transfected cells were then subjected to a wound-healing assay as described under “Experimental procedures.” Representative images of cells at 0 and 18 h postscratching were shown. Transfected cells stained with the anti-GFP antibody were also shown. B, percentage of wound closure as described in A was quantified. Data are summarized from three independent experiments. V, vector-transfected control. Error bars, S.D.

Figure 5.

Lys-42 is essential for mediating invasion of transformed cells. A, Tet293 cells stably transfected with K-Ras^V12 or K-Ras^V12/R42 expression plasmid were cultured in the presence of Dox for 24 h, after which treated cells, along with parental cells, were used for transwell migration assays. Representative images are shown. B, cell invasiveness as described in A was quantified. Data are summarized from three independent experiments. Ve, vector-transfected control. C, Tet293 cells stably transfected with a plasmid construct expressing K-Ras^V12 or K-Ras^V12/R42 were cultured in the presence of Dox for various times as indicated, after which cells were collected and lysed. Equal amounts of cell lysates were blotted with antibodies to Snail and β-actin, respectively. Molecular weight markers are also indicated. *, p < 0.05. Error bars, S.D.

Figure 6.

Expression of Lys-42 mutant of K-Ras compromises invasion of nontransformed cells. A, NIH3T3 cells were transfected with a plasmid construct expressing K-Ras^V12 or K-Ras^V12/R42 for 24 h, after which transfected cells, along with cells transfected with vector alone, were subjected to transwell migration assays. Representative images are shown. B, cell invasiveness as described in A was quantified. Data are summarized from three independent experiments. *, p < 0.05. Error bars, S.D.

Figure 7.

SUMO inhibitor blocks migration of pancreatic cancer cells with K-Ras^V12 mutation. A, both BxPC-3 (without K-Ras mutation) and MiaPaCa-2 (with K-Ras^V12 mutation) cells were employed for wound-healing assays. Cells with open scratches were treated with various concentrations of 2-D08 for 18 h. Representative images of cells before (CNTL) and after recovery in the presence of 2-D08 for 18 h are shown. B, cell migration of various treatments, as shown in A, was quantified. Data are summarized from three independent experiments. C, pancreatic cell lines as indicated were immunoprecipitated with the Ras antibody (Pan-Ras) or IgG as control. Immunoprecipitates, along with lysate controls, were blotted with antibodies to SUMO2/3 or to K-Ras. D, MiaPaCa-2 cells treated with or without 2-D08 were lysed, and equal amounts of cell lysates were blotted with antibodies to N-cadherin (N-Cad), ZEB1, ZO-1, PARP-1, and β-actin, respectively. Molecular weight markers are also indicated.

Figure 8.

A model depicting sumoylation in regulating Ras signaling and functions. SIM, SUMO-interacting motif; RBD, Ras-binding domain.