Rab32 regulates Golgi structure and cell migration through Protein Kinase A-mediated phosphorylation of Optineurin

Abstract

Rab32 is a small GTPase and molecular switch implicated in vesicular trafficking. Rab32 is also an A-Kinase Anchoring Protein (AKAP), which anchors cAMP-dependent Protein Kinase (PKA) to specific subcellular locations and specifies PKA phosphorylation of nearby substrates. Surprisingly, we found that a form of Rab32 deficient in PKA binding (Rab32 L188P) relocalized away from the Golgi apparatus and induced a marked disruption in Golgi organization, assembly, and dynamics. Although Rab32 L188P did not cause a global defect in PKA activity, our data indicate that Rab32 facilitates the phosphorylation of a specific PKA substrate. We uncovered a direct interaction between Rab32 and the adaptor protein optineurin (OPTN), which regulates Golgi dynamics. Further, our data indicate that optineurin is phosphorylated by PKA at Ser342 in a Rab32-dependent manner. Critically, blocking phosphorylation at OPTN Ser342 leads to Golgi fragmentation, and a phospho-mimetic version of OPTN rescues Golgi defects induced by Rab32 L188P. Finally, Rab32 AKAP function and OPTN phosphorylation are required for Golgi repositioning during cell migration, contributing to tumor cell invasion. Together, these data reveal a role for Rab32 in regulating Golgi dynamics through PKA-mediated phosphorylation of OPTN.

Keywords: Golgi; Optineurin; Protein Kinase A; Rab32; migration.

Fig. 1.

The AKAP binding mutant of Rab32 mislocalizes away from the Golgi and disrupts Golgi morphology. (A) Huh7 cells transfected with Flag-Rab32 WT or L188P (green) were stained with the Golgi marker GM130 (magenta). (B) Quantitation of overlap between Flag-Rab32 and GM130 reflects the marked decrease exhibited by Rab32 L188P. (C) Representative images showing a normal Golgi morphology in cells expressing Rab32 WT versus a disrupted Golgi phenotype in cells expressing Rab32 L188P. (D) Categorization of the Golgi morphology in cells transfected with the indicated forms of Rab32. n = 10 fields per condition in each of three independent biological replicates. (E) Knockdown of Rab32 alters Golgi morphology. Representative images of Huh7 cells depleted of Rab32 and stained for GM130 (green). (F) Categorization of the Golgi morphology in cells transfected Rab32 siRNA versus nontargeting (NT) control siRNA. (G) Rab32 GTPase activity supports Golgi morphology. Huh7 cells transfected with GFP-Rab32 Q85L or T39N (green) were stained for the Golgi protein GM130 (magenta). (H) Categorization of the Golgi morphology in cells transfected with the indicated forms of Rab32. n = 10 fields per condition in each of three independent biological replicates. (I) Representative images showing examples of Golgi (GM130, magenta) with the indicated morphologies used in D, F, and H. Graphs indicate the mean ± SD of three independent biological replicates. Statistical significance was determined by Student’s t test. * P < 0.05, ** P < 0.01. (Scale bars in low magnification images = 10 µm; in magnified images = 1 µm.)

Fig. 2.

Rab32 promotes Golgi assembly. (A) Huh7 cells were transfected with Flag-Rab32 WT or L188P and incubated with Brefeldin A (BFA) for 1 h. The BFA was then washed out and cells were fixed at the indicated timepoints and stained for Flag-Rab32 (green) or GM130 (magenta). (B) Quantitation of the Golgi fragment area revealed smaller fragments and delayed reassembly in cells expressing Rab32 L188P (n = 20 cells per condition in each experiment). Gray bars: Rab32 WT, white bars: Rab32 L188P. (C) Huh7 cells were depleted of Rab32 using siRNA, subjected to BFA washout as in A, and stained for GM130 (magenta). (D) Quantitation of the Golgi fragment area (n = 15 to 35 cells per condition in each experiment). Gray bars: nontargeting siRNA, white bars: Rab32 siRNA. (E) Huh7 cells were transfected with GFP-Rab32 WT, Q85L, or T39N (green), subjected to BFA washout as in A, and stained for GM130 (magenta). (F) Quantitation of the Golgi fragment area (n = 10 fields per condition in each experiment). Dark gray bars: Rab32 WT, light gray bars: Rab32 Q85L, white bars: Rab32 T39N. Graphs indicate the mean ± SD of three independent biological replicates. Statistical significance was determined by Student’s t test. *P < 0.05, ***P < 0.001, ns = not statistically significant. (Scale bar, 10 µm.)

Fig. 3.

A novel interaction between Rab32 and OPTN. (A) GFP-Trap pulldown of lysates from Huh7 cells transfected with GFP or the indicated forms of GFP-Rab32 and immunoblotted for OPTN. (B) Purified His-OPTN was incubated with the indicated forms of GST-Rab32, and the resulting GST pulldowns were immunoblotted for His-OPTN to indicate a direct interaction. (C) Huh7 cells were transfected with Flag-tagged Rab32 WT or L188P and stained for endogenous OPTN. (D) Quantitation of the overlap between OPTN and Flag-Rab32 WT/L188P shows similar colocalization. Manders coefficient, n = 20 cells per experiment. (E) Domain structure of OPTN, indicating the predicted consensus PKA phosphorylation site in the Coiled Coil domain (CC). Ser342 (red, bold) is preceded by two basic amino acids at the −2 and −3 position (red). (F and G) Phosphorylation of endogenous OPTN. (F) Huh7 cells were treated with 10 µM FSK to activate cAMP-dependent PKA, and lysates were immunoprecipitated using the phospho-PKA substrate antibody and immunoblotted for OPTN. (G) Huh7 cells were treated with FSK, lysates were immunoprecipitated for OPTN, and immunoblotted with a phospho-PKA substrate antibody. (H) Huh7 cells transfected to overexpress mCherry vector or mCherry OPTN were treated with the cAMP activator FSK (10 µM, 10 min) and PKA inhibitor H89 (40 µM, 2 h). Lysates were immunoprecipitated with an anti-mCherry antibody and immunoblotted with a phospho-PKA substrate antibody. The PKA inhibitor H89 substantially diminished FSK-stimulated OPTN phosphorylation. (I) Huh7 cells transfected to overexpress mCherry vector, OPTN WT, or OPTN S342A were stimulated with FSK (10 µM, 10 min), immunoprecipitated, and immunoblotted as in H. The S342A mutation markedly reduced OPTN phosphorylation. (J) Rab32 is required for OPTN phosphorylation. Huh7 cells depleted of Rab32 by siRNA were treated with FSK (10 µM, 10 min), then immunoprecipitated for OPTN, and immunoblotted with a phospho-PKA substrate antibody. Phospho-OPTN was normalized to total OPTN. All immunoblots are representative of at least three independent biological replicates. Graphs represent mean ± SD of three independent biological replicates. **P < 0.01, ns: not statistically significant. (Scale bars in low magnification images = 10 µm; in magnified images = 1 µm.)

Fig. 4.

OPTN phosphorylation by PKA maintains Golgi integrity to support cell migration. (A) Huh7 cells were transfected with mCherry-tagged OPTN WT or S342A (pseudocolored in green) and stained for the Golgi protein GM130 (magenta). (B) Quantitation of the percent of cells with the indicated Golgi morphology shows increased fragmentation in mutant-expressing cells. n = 10 fields per experiment in each of three independent biological replicates. (C) A phospho-mimetic form of OPTN rescues Rab32 L188P-mediated Golgi defects. Huh7 cells were transfected with Flag-Rab32 WT or L188P (blue) and mCherry OPTN WT or S342A (pseudocolored green) and stained for GM130 (magenta). (D) Quantitation of the percent of cells with the indicated Golgi morphology. n = 10 fields per experiment in each of three independent biological replicates. (E) A phospho-mimetic form of OPTN rescues Golgi assembly following BFA washout. Huh7 cells were transfected as in D, treated with Brefeldin A for 1 h, then washed out for 3 h. (F) The area of the Golgi fragments following BFA washout was measured and is normalized to the cells expressing Rab32 WT and OPTN WT. (G) Huh7 cells transfected with Flag-Rab32 WT or L188P were seeded in a wound healing migration assay. 4 h after scratching the monolayer, the cells were fixed and stained for the Golgi protein GM130 and Flag-Rab32 (dashed line indicates transfected cells). The arrow indicates the direction of migration. (H) Quantitation of the percent of cells with the Golgi in the indicated position relative to the nucleus and relative to the direction of cell migration. (I) Huh7 cells transfected with mCherry OPTN WT or S342A and seeded in a wound healing assay as in G. (J) Quantitation of the percent of cells with the Golgi in the indicated position relative to the nucleus and relative to the direction of cell migration. (K) Huh7 cells were co-transfected with GFP-Rab32 L188P and either mCherry OPTN WT or S342E and seeded in a wound healing assay as in G. Quantitation of the percent of cells with the Golgi in the indicated position relative to the nucleus and relative to the direction of cell migration. In H, J, and K, n = 88 to 110 cells per condition were scored in each of three independent biological replicates, and a Student’s t test was used to compare the percent of cells with “front” orientation of the Golgi. (L) Huh7 cells transfected with siRNA to Rab32 or a control siRNA were seeded in a transwell assay. A representative immunoblot and quantitation of three independent replicates demonstrate the degree of Rab32 knockdown. Huh7 cells were transfected with GFP-Rab32 WT or L188P (M) or mCherry-OPTN WT or S342A (N) and seeded in a transwell assay. After 24 h, the percent of cell invasion was scored. Graphs indicate the mean ± SD of three independent biological replicates. Statistical significance was determined by Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001 ****P < 0.0001, ns = not statistically significant. (Scale bars in low magnification images = 10 µm; in magnified images = 1 µm.) (O) Model: 1) The AKAP Rab32 promotes PKA-mediated phosphorylation of OPTN to regulate Golgi dynamics and assembly during cell migration. 2) Disrupting this phosphorylation pathway by depleting Rab32, locking Rab32 in the GDP-bound state to decrease OPTN binding (T39N mutation), or blocking Rab32 binding to PKA (L188P mutation), disrupts Golgi morphology, and impairs Golgi dynamics and assembly.