Phosphorylation of G protein-coupled receptor kinase 2-interacting protein 1 tyrosine 392 is required for phospholipase C-gamma activation and podosome formation in vascular smooth muscle cells

Abstract

Objective: Podosomes, which are actin-rich structures, contribute to cell motility, matrix remodeling, and tissue remodeling. We have shown that G protein-coupled receptor kinase 2-interacting protein 1 (GIT1) colocalizes with podosomes and is important in podosome formation in endothelial cells. Src stimulates GIT1 tyrosine phosphorylation, which is critical for phospholipase C-γ (PLCγ) activation. In this study, we identified specific GIT1 tyrosines required for PLCγ activation and podosome formation in vascular smooth muscle cells (VSMC).

Methods and results: We used phorbol 12,13-dibutyrate (PDBU) to induce podosomes in A7r5 VSMC. GIT1 colocalized with podosomes and GIT1 knockdown using short interfering RNA significantly reduced podosome formation. PDBU stimulated GIT1 tyrosine phosphorylation. GIT1 tyrosine phosphorylation was dramatically decreased in SYF-/- cells, and it was also reduced by pretreatment with the protein kinase C (PKC) and Src inhibitors, suggesting that GIT1 phosphorylation was dependent on PKC and Src. By mutation analysis of multiple tyrosines, we found that PDBU specifically increased GIT1-Y392 phosphorylation. Overexpression of GIT1 (Y392F) but not of GIT1 (Y321F) decreased PDBU-mediated PLCγ activation and podosome formation without effect on extracellular signal-regulated kinase 1/2 activation. Additionally, we provide evidence that GIT1 knockout VSMC have markedly fewer podosomes on PDBU treatment compared with wild-type VSMC. These data show that GIT1 is a key regulator of podosome formation in VSMC.

Conclusions: In conclusion, our data suggest that GIT1-Y392 phosphorylation is critical for PDBU-induced podosome formation by regulating PLCγ activation. We propose that specific signaling modules are assembled in a GIT1 phosphotyrosine-dependent manner as exemplified by PLCγ activation versus extracellular signal-regulated kinase 1/2 activation.

Figure 1. Role of GIT1 in PDBU-induced podosome formation

(A) Representative immunofluorescent images showing F-actin (Phalloidin) and GIT1 localization in podosomes. A7r5 cells were starved for 6 hours and then treated with or without 1μM PDBU for 60 min. Cells were fixed with 4% formaldehyde and double-stained for either F-actin with TRITC-Phalloidin and GIT1. F-actin is in red and GIT1 is in green. Panels c and f are the merged images. Arrows show podosomes. Insets are enlarged images of podosome-enriched areas. (B) Effects of GIT1 siRNA on GIT1 expression. A7r5 cells were transiently transfected with either 100nM control siRNA or GIT1 siRNA per dish. After 48 hours, cell lysates were immunoblotted with anti-GIT1 antibody. Equal loading was confirmed by β-actin. (C) Effect of GIT1 siRNA on podosome formation. Podosomes were characterized and quantified as described in Methods. The data are expressed as percentage (%) of podosome positive cells (n=100 cells per dish). All values are expressed as mean ± SD of three independent experiments performed in triplicate. A p value < 0.05 was considered to be significant (*). (D) GIT1 WT and KO VSMC were cultured for 48 hrs, serum starved overnight and treated vehicle (DMSO) or PDBU for 4hrs. Cells were fixed and stained with TRITC-Phalloidin to identify podosomes (arrows).

Figure 2. Role of PKC and Src in PDBU-induced GIT1 phosphorylation

PDBU-induced GIT1 tyrosine phosphorylation in A7r5 (A) and in HEK293 (B) cells. Flag-GIT1 (WT) was transfected into A7r5 or HEK293 cells for 24 hours. The cells were starved for 6 hours prior to being treated with or without 1μM PDBU for various times. (C) Effect of PKC and Src inhibition on PDBU-induced GIT1 tyrosine phosphorylation. After transfection with Flag-GIT1 (WT), HEK293 cells were pretreated with 10 μM PP2 (Src inhibitor) or 5 μM Go6850 (PKC inhibitor) for 30 min and then treated with or without 1μM PDBU for 10 min. (D) Role of Src in PDBU-induced GIT1 tyrosine phosphorylation. SYF+/+ and SYF-/- fibroblasts were infected with Flag-GIT1 (WT) adenovirus for 24 hours and then starved for 6 hours followed by treatment with or without 1μM PDBU for 10 min. Lysates were immunoprecipitated with GIT1 antibody and then immunoblotted with 4G10 antibody to detect pGIT1 (top panels). To confirm equal protein immunoprecipitation, the blot was reprobed with GIT1 antibody (lower panels). The blots were analyzed by densitometry using LiCor software. Fold changes normalized to 0 min (A-B) or to the first lane as shown below each blot (n=2-3).

Figure 3. Role of GIT1-Y293, -Y321, and –Y392 in PDBU-induced GIT1 tyrosine phosphorylation and podosome formation

Effects of GIT1 tyrosine mutants on PDBU-induced GIT1 tyrosine phosphorylation. HEK293 cells were transfected with Flag-GIT1 (WT), Flag-GIT1 (Y293F), and Flag-GIT1 (Y392F) (A) or Xpress-GIT1 (WT) or Xpress-GIT1 (Y321F) (B) for 24 hours. Cells were starved for 6 hours and then treated with or without 1μM PDBU for 10 min. Cell lysates were immunoprecipitated with Flag antibody or Xpress antibody, then immunoblotted using 4G10 antibody to detect pGIT1 (top panel), and reprobed to detect GIT1 (lower panel). The blots were analyzed by densitometry using LiCor software. Fold changes normalized to the first lane are shown below the blots (n=2-3). Effects of GIT1 tyrosine mutants on PDBU-induced podosome formation. A7r5 cells were transfected with different GIT1 mutants for 24 hours followed by treatment with or without 1μM PDBU for 60 min. Cells were stained for GIT1 and podosomes. Percentage of podosome positive cells (%) were counted and analyzed only in cells expressing GIT1 mutant (with green fluorescence). Quantified data are presented as percentages of podosome positive cells in GIT1 mutant positive cells (n=100 cells per dish). All values are expressed as mean ± SD of three independent experiments performed in triplicate. A p value < 0.05 was considered to be statistically significant (*)

Figure 4. Role of PLCγ in PDBU-induced podosome formation

(A) PDBU induced PLCγ phosphorylation. Flag-GIT1 and PLCγ were co-transfected into HEK293 cells for 24 hours. Cells were starved for 6 hours and treated with or without 1μM PDBU for the indicated times. Cell lysates were immunoprecipitated with PLCγ antibody and then immunoblotted with pPLCγ (Y783) antibody and reprobed with PLCγ antibody. The total cell lysates (TCL) were detected with Flag antibody to show the Flag-GIT1 expression. Equal loading was confirmed by immunoblotting for β-actin. The blots were analyzed by densitometry using LiCor software. Fold changes normalized to the first lane are shown below the blots (n=2-3). (B and C) Effects of PLCγ inhibitor (U73122) on podosome formation. A7r5 cells were pretreated with 5μM U73122 for 30 min prior to treatment with or without 1μM PDBU for 60 min. Cells were then stained for podosomes (B). Quantified data are presented as percentages of podosome positive cells (n=100 cells per dish) (C). All values are expressed as mean ± SD of three independent experiments performed in triplicate. A p value < 0.05 was considered to be significant (*)

Figure 5. Role of GIT1 tyrosine phosphorylation in PLCγ activation and GIT1-PLCγ interaction

(A) Effects of GIT1 tyrosine mutants on PLCγ activation. HEK293 cells were co-transfected with PLCγ together with pCDNA3 vector, Flag-GIT1 (WT), Flag-GIT1 (Y293F), or Flag-GIT1 (Y392F) (B) for 24 hours and then starved for 6 hours. Cells were treated with or without 1μM PDBU for 10 min and cell lysates were immunoprecipitated with PLCγ antibody and immunoblotted with pPLCγ antibody (top panel). To confirm equal protein immunoprecipitation, the blot was reprobed with PLCγ antibody (lower panel). (B) Effects of GIT1 tyrosine mutants on GIT1-PLCγ interaction. HEK293 cells were co-transfected with PLCγ together with Flag-GIT1 (WT), Flag-GIT1 (Y293F), or Flag-GIT1 (Y392F) for 24 hours and starved for 6 hours. After treatment with 1μM PDBU for 10 min, cell lysates were immunoprecipitated with Flag antibody for GIT1 and then immunoblotted with PLCγ antibody. To confirm equal protein immunoprecipitation, the blot was reprobed with GIT1 antibody. The blots were analyzed by densitometry using LiCor software. Fold changes normalized to the first lane are shown below the blots (n=2-3).

Figure 6. Role of GIT1 tyrosine phosphorylation in PDBU-induced ERK1/2 activation

HEK 293 cells were transfected with (A) pcDNA3, Flag-GIT1 (WT), Flag-GIT1 (Y293F), or Flag-GIT1 (Y392F) or (B) pcDNA3, Xpress-GIT1 (WT), or Xpress-GIT1 (Y321F) for 24 hours and starved for 6 hours. After treatment with or without 1μM PDBU for 10 min, cell lysates were immunoblotted with specific pERK1/2 antibody (upper panels) and reprobed with ERK2 antibody (lower panels). Flag (or Xpress) antibody blot shows the expression of various GIT1 plasmids, and β-actin antibody blot shows equal loading. The blots were analyzed by densitometry using LiCor software. Fold changes normalized to the first lane are shown below the blots (n=2-3).