Protein Kinase C-αααα and Protein Kinase C-εεεε are Required for Grb2-associated Binder-1 Tyrosine-phosphorylation in Response to Platelet-derived Growth Factor

Statistical


Introduction
The members of the insulin receptor substrate (IRS) family participate in signaling cascades activated by various cytokines (1,2).These proteins have multiple tyrosinephosphorylation sites that act as docking sites for the Src homology (SH) 2 domains of various signaling proteins.These allow IRS proteins to function as multi-site docking proteins and link growth factor receptors to multiple signaling pathways.Gab1 is structurally similar to the Drosophila daughter of sevenless protein (DOS) and to IRS-1 with an amino-terminal pleckstrin homology (PH) domain, several proline-rich sequences, and multiple potential tyrosinephosphorylation sites for binding of SH2 and SH3 containing proteins (3)(4)(5).Gab1 is rapidly phosphorylated on tyrosine residues upon stimulation of cells with EGF, insulin, hepatocyte growth factor (HGF), nerve growth factor, interleukin (IL)-3, IL-6, and erythropoietin (6)(7)(8)(9)(10).
To date, several signaling molecules, such as the adapter protein Grb2, the proteintyrosine phosphatase SHP2, p85 phosphatidylinositol 3-kinase (PI3-K), and PLCγ, have been found to associate with Gab1 in various cell lines (6,8,9,11,12).However, it has not yet been established what kinase is responsible for Gab1 tyrosine-phosphorylation or whether Gab1 is required for growth factor mediated signal transduction.In the present study, we demonstrated that Gab1 was rapidly tyrosine-phosphorylated by PDGF in VSMC.We then investigated the upstream mediators of Gab1 phosphorylation.Based on the association between Gab1 and PLCγ (11,12), we investigated the potential role of PLCγ for Gab1 tyrosine-phosphorylation.We used CHO cells expressing mouse wild type (WT) and mutant (Y977/989F: lacking the PLCγ binding site) PDGFβ receptor and the specific PLCγ inhibitor U73122.Gab1 tyrosinephosphorylation was decreased in both Y977/989F transfected CHO cell and VSMC pretreated with U73122.by guest on March 24, 2020 http://www.jbc.org/Downloaded from PKC is a molecule activated downstream of PLCγ, and Gab1 tyrosine-phosphorylation by PDGF was totally blocked after inhibiting PKC activity.Experiments using phorbol esters suggested the involvement of conventional PKC (cPKC) and/or novel PKC (nPKC) for this process.We previously demonstrated the expression of PKC-α (cPKC), PKC-δ and PKCε (nPKC) in cultured VSMC (13).Studies with PKC inhibitors and antisense PKC oligonucleotides showed that PKC-α and PKC-ε, but not PKC-δ, are required for Gab1 tyrosinephosphorylation by PDGF.Gab1 tyrosine-phosphorylation was inhibited by protein tyrosine kinase inhibitor genistein, showing importance of genistein-sensitive tyrosine kinases in this signaling event.
It has been suggested that tyrosine-phosphorylated Gab1 associates with other signaling molecules such as SHP2 and phosphatidyl inositol 3-kinase (PI3-K), and subsequently regulates mitogen-activated protein kinase (MAPK) activity (9,(14)(15)(16)(17)(18).In the present study, PDGFinduced Gab1 tyrosine-phosphorylation increased the association with SHP2 and PI3-K.The binding between Gab1 and SHP2 or PI3-K was decreased by PLCγ and/or PKC inhibition that was parallel to the abrogation of Gab1 tyrosine-phosphorylation.Since PDGF-mediated ERK activation was significantly enhanced in Gab1-overexpressed CHO cells, these results indicate the importance of the PLCγ/PKC dependent Gab1 tyrosine-phosphorylation for the interaction with other signaling molecules, which subsequently regulates ERK activation.
Recombinant human EGF was from Clonetics (San Diego, CA, USA).
Cell Culture -Rat aortic VSMC were isolated from the thoracic aorta of 200-250 g male Sprague-Dawley rats and maintained in DMEM supplemented with 10% serum as described (19).VSMC of passage 8 to 14 at 70-80% confluence were growth-arrested by incubation in DMEM without serum for 48 hours before use.Mouse PDGFβ receptor wild type (WT) or mutant (Y977/989F) expressing Chinese hamster ovary (CHO) cells were a kind gift from Dr.

RESULTS
EGF and PDGF Stimulate Gab1 tyrosine-phosphorylation -To measure the effect of growth factors on Gab1 tyrosine-phosphorylation, VSMC were exposed to 10 ng/ml EGF or PDGF-BB for 1 to 20 minutes.After the preparation of cell lysates, Gab1 was immunoprecipitated and immunoblots were performed for phosphotyrosine.Tyrosine-phosphorylation of Gab1 occurred within 1 minute and peaked at 3 and 5 minutes after exposure to EGF and PDGF respectively (Fig. 1).
It has been reported that cross-talk (transactivation) exists among growth factor receptors (21,22).To determine the role of PDGF-induced EGF receptor transactivation in Gab1 tyrosinephoshorylation, AG compound studies were performed (Fig. 2).VSMC were preincubated with 10 µM AG1295 and AG1296 (specific PDGF receptor kinase inhibitors), or AG1478 (specific EGF receptor kinase inhibitor) for 30 minutes before EGF and PDGF stimulation.We measured the inhibitor effects of AG compounds by their effect on tyrosine-phosphorylation in coimmunoprecipitated EGF receptor (170 kDa) or PDGF receptor (180 kDa).Gab1 tyrosinephosphorylation induced by EGF was inhibited by AG1478, but not by AG1295 or AG1296.On the contrary, PDGF-induced Gab1 tyrosine-phosphorylation was inhibited by AG1295 and AG1296, but not by AG1478.These results show that EGF receptor kinase activity is required for Gab1 tyrosine-phosphorylation when VSMC were stimulated by EGF, but transactivated EGF receptor kinase activity is not required when stimulated by PDGF.Inhibition of PDGFinduced Gab1 tyrosine-phosphorylation with AG1295 and AG1296 suggests that PDGF receptor kinase activity is responsible for this signaling event.
by guest on March 24, 2020 http://www.jbc.org/Downloaded from Role of PLCγ in PDGF-induced Gab1 phosphorylation -Although PLCγ has been reported to be associated with Gab1 (11,12), the function of this association has not yet been elucidated.To investigate the role of PLCγ in Gab1 tyrosine-phosphorylation, we prepared CHO cells transfected with mouse PDGFβ receptor wild type (PDGFβR-WT) or mutant (PDGFβR-Y977/989F) which is unable to interact with PLCγ.In transfected CHO cells stimulated by PDGF (10 ng/ml, 5 minutes), both PDGFβR-WT and PDGFβR-Y977/989F were tyrosinephosphorylated to the same extent (Fig. 3A).However, Gab1 tyrosine-phosphorylation by PDGF was significantly impaired in CHO cells transfected with PDGFβR-Y977/989F compared to PDGFβR-WT, demonstrationg PDGFβ receptor kinase activity (Fig. 3B).PDGF-induced Gab1 tyrosine-phosphorylation was also abrogated after pretreatment with the PLCγ inhibitor 10 µM U73122 (Fig. 3C).These results indicate that PLCγ activity plays an important role in Gab1 tyrosine-phosphorylation induced by PDGF.The results further suggest that the PDGFβ receptor is not the Gab1 tyrosine kinase.

PKC activity is required for Gab1 tyrosine-phosphorylation and ERK activation -Because PLCγ
stimulates diacylglycerol-dependent PKC isoforms, we further investigated the role of PKC in PDGF-stimulated Gab1 tyrosine-phosphorylation.To examine the signaling pathway downstream of PKC, we treated VSMC with the phorbol esters PMA and PDBU, which bind and activate PKC (cPKC and nPKC).Gab1 tyrosine-phosphorylation was stimulated by 200 nM PMA to the same extent as by PDGF (Fig. 4, compare lanes 2 and 3).
Longer exposure to phorbol ester is known to down-regulate PKC expression (13).
VSMC were pretreated with 1 µM PDBU for 24 hours to down-regulate phorbol ester-responsive PKC isoforms, and stimulated with PDGF.As shown in Fig. 4 (lane 4), PDGF-mediated Gab1 by guest on March 24, 2020 http://www.jbc.org/Downloaded from tyrosine-phosphorylation was significantly inhibited in PDBU treated cells.These findings suggest a significant role for conventional and/or novel PKC isoforms for Gab1 tyrosinephosphorylation.
It has been reported that PKC is essential for erythropoietin receptor tyrosinephosphorylation and regulates Gab1 tyrosine-phosphorylation by erythropoietin (23).To investigate the possibility that PDGFβ receptor is positively regulated by PKC in this context, we treated VSMC with chelerythrine (PKC inhititor acting on the catalytic domain).Although PDGF-induced Gab1 tyrosine-phosphorylation was significantly inhibited by chelerythrine pretreatment (Fig. 4B), tyrosine-phosphorylation in co-immunoprecipitated PDGFβ receptor was not inhibited, suggesting PKC regulates Gab1 tyrosine-phosphorylation downstream of PDGFβ receptor but not by regulating the tyrosine-phosphorylation status of PDGFβ receptor itself.It is also suggested that the binding between PDGFβ receptor and Gab1 is independent of the tyrosine-phosphorylation status of Gab1.
PKC-α and PKC-ε are required for Gab1 tyrosine-phosphorylation -We have previously characterized PKC isozyme expression in cultured VSMC, and showed that PKC-α, PKC-δ and PKC-ε are expressed among cPKC and nPKC (13).To determine the PKC isozyme specificity for Gab1 tyrosine-phosphorylation, we incubated VSMC with rottlerin, which is a specific PKCδ inhibitor, and stimulated with PDGF.As shown in Fig. 5A, PDGF-induced Gab1 tyrosinephosphorylation was not blocked by rottlerin pretreatment.Next, we used antisense PKC-α and PKC-ε oligonucleotides.CHO cells were stimulated with PDGF 72 hours after transfection.
Expression of PKC-α and PKC-ε were effectively decreased (Fig. 5  VSMC were pretreated with general PTK inhibitors, herbimycin A and genistein, and stimulated by PDGF.As shown in Fig. 6A, Gab1 tyrosine-phosphorylation was significantly inhibited by genistein but not by herbimycin A. Herbimycin A is known to show inhibitory activity preferably to Src related kinases.Src family kinase inhibitor PP2 also failed to inhibit Gab1 tyrosine-phosphorylation by PDGF (Fig. 6B).These results show that Gab1 tyrosinephosphorylation is regulated by genistein-sensitive kinases different from Src family kinases.

Effect of Gab1 overexpression on PDGF-induced ERK activation -To investigate the role of
Gab1 in signaling pathways downstream of PDGFβ receptor, we prepared CHO cells expressing PDGFβR-WT that were co-transfected with a hemagglutinin (HA)-epitope tagged ERK2 and with a HA-tagged Gab1.PDGF stimulation of ERK2 phosphorylation was significantly increased (p < 0.001) in CHO cells that overexpress Gab1 as compared with control (vector) cells (Fig. 7A).

DISCUSSION
The major findings of this study are that Gab1 is tyrosine-phosphorylated by PDGF via a pathway that requires PLCγ, PKC-α and PKC-ε activity, and genistein-sensitive tyrosine kinases (Fig. 8).Gab1 is important for PDGF signaling since overexpression leads to enhanced ERK activation upon stimulation by PDGF (Fig. 7A).
It has been reported that Gab1 is tyrosine-phosphorylated downstream of EGF receptor (6,24,25).In this report, we demonstrated that Gab1 is also tyrosine-phosphorylated by PDGF in VSMC.Because there is accumulating evidence that EGFR is transactivated by PDGF (21,26), there was a possibility that Gab1 was tyrosine-phosphorylated downstream of EGFR that was transactivated by PDGF.In fact, Daub et al. showed that Gab1 is tyrosine-phosphorylated downstream of transactivated EGFR in COS-7 cells when stimulated with G-protein coupled receptor agonist (24).We successfully excluded this possibility because AG1478 (a specific EGFR kinase inhibitor) failed to inhibit PDGF-induced Gab1 tyrosine-phosphorylation.
Moreover, a direct association between Gab1 and PDGF receptor was demonstrated because PDGF receptor was co-immunoprecipitated by anti-Gab1 (Fig. 1 and 2).
To characterize signaling molecules that are involved in Gab1 tyrosine-phosphorylation, we first investigated PLCγ which was already reported to be associated with Gab1 (11,12).Our study demonstrated PDGF-induced Gab1 tyrosine-phosphorylation was impaired in PDGFβ receptor mutant (Y977/989F: lacking biding sites for PLCγ) expressing CHO cells as compared to PDGFβ receptor wild type.The PLCγ inhibitor (U73122) dramatically reduced Gab1 tyrosinephosphorylation induced by PDGF.These results strongly suggest that PLCγ and its downstream signaling cascade (but not PDGFβ receptor tyrosine kinase activity) play an important role in Gab1 tyrosine-phosphorylation. by guest on March 24, 2020 http://www.jbc.org/Downloaded from PKC isozymes are serine-threonine kinases activated by diacylglycerol (DG), which is a product generated by PLCγ.PKC consists of three subtypes, cPKC, nPKC and atypical PKC.
Our data using PMA and PDBU suggested that phorbol ester-responsive PKC isoforms (cPKC and nPKC) may be responsible for Gab1 tyrosine-phosphorylation.In a previous report, we found PKC-α (cPKC), PKC-δ and PKC-ε (nPKC) are expressed predominantly in cultured VSMC (13).There is accumulating evidence that PKC-α and PKC-ε are involved in important VSMC functions.Haller et al observed these two PKC isoforms were translocated to focal adhesions during integrin-induced VSMC spreading, and inhibition of these PKC isoforms with antisense oligonucleotides significantly inhibited cell spreading (27).Giardina et al demonstrated that oxidized-LDL enhanced coronary vasoconstriction by increasing the activity of PKC-α and PKC-ε (28).In the present study, our results showed the involvement of PKC-α and PKC-ε in PDGF-induced Gab1 tyrosine-phosphorylation.PKC-δ was considered unlikely based on experiments using rottlerin (a PKC-δ inhibitor).However, the potential lack of specificity and cell-specific ineffectiveness of drug inhibitors such as rottlerin limits our ability to completely rule out PKC-δ or other PKC isozymes.Further study with other independent approaches will be required to define the precise role of PKC isozymes.
The ubiquitously expressed Src family kinase members seem to be important mediators of PDGFβ receptor signal events, regulating the mitogenic response to PDGF (29).We also previously reported that Src activity was important for ERK activation through transactivated EGF receptor by PDGF (22).However, the present study showed no evidence for Src family kinase involvement in Gab1 tyrosine-phosphorylation because herbimycin A and PP2 failed to inhibit this signaling event.Genistein successfully inhibited PDGF-induced Gab1 tyrosine-by guest on March 24, 2020 http://www.jbc.org/Downloaded from phosphorylation.Further study is required to identify the particular tyrosine kinases that phosphorylate Gab1.
In this study, we observed that overexpression of Gab1 in CHO cells significantly enhanced ERK activation by PDGF, suggesting that Gab1 plays an important role for ERK activation downstream of PDGFβ receptor in VSMC.Although the mechanism by which Gab1 contributes to ERK activation is still not fully understood, it has been reported that Gab1 needs to be tyrosine-phosphorylated to interact with other signaling molecules.For example, SHP2 and PI3-K bind to Gab1 that is tyrosine-phosphorylated by insulin ( 6), IL-6 (9,14), EGF (15)(16)(17)25,30), erythropoietin (10,31), NGF (8,32) and HGF (4,7,18).The activation of the ERK members of the MAPK family are regulated by SHP2 and PI3-K bound to tyrosinephosphorylated Gab1 (9,14-18).Moreover, SHP2 and PI3-K activation mediated by Gab1 tyrosine-phosphorylation has been suggested to promote cell survival (8), DNA synthesis, cell differentiation (32), and Gab1 localization to the plasma membrane (33)(34)(35).Itoh et al reported that Gab1-deficient mice are embryonic-lethal and display developmental defects in the heart, placenta, and skin, similar to phenotypes observed in mice lacking signals mediated by the HGF, PDGF and EGF pathways.Because ERK is activated at much lower levels in cells from Gab1deficient embryos in response to these growth factors or cytokines, these findings suggest that Gab1 is a common player in a broad range of growth factor and cytokine signaling pathways linking ERK MAPK activation (14).In the present study, we demonstrated that the binding of SHP2 and PI3-K to Gab1 was increased in proportion to Gab1 tyrosine phosphorylation induced by PDGF in VSMC.A significant reduction in SHP2 and PI3-K associated with Gab1 was observed when Gab1 tyrosine-phosphorylation was inhibited by the blocking PLCγ/PKC activity.Considering that the expression of Gab1 protein is not affected by 5 minute-PDGF       Serum-starved VSMC were stimulated with 10 ng/ml PDGF-BB for 5 minutes after preincubation with 50 µM genistein (Gen) or 10 µM herbimycin A (Herb) (A), or 1 µM PP2 (B) for 30 min.Cell lysates were immunoprecipitated with anti-Gab1 followed by immunoblot with 4G10 (upper panel), and reprobed with anti-Gab1 (lower panel).
by guest on March 24, 2020 http://www.jbc.org/Downloaded from incubation in VSMC, our results suggest that Gab1 tyrosine-phosphorylation is an important factor to regulate the PDGF-induced ERK activation, interacting with other molecules including SHP2 and PI3-K.In summary, PDGF-mediated ERK activation is positively regulated by tyrosinephosphorylated Gab1, which is also positively controlled by two different PKC isoforms, PKC-α and PKC-ε, when VSMC are stimulated with PDGF.We conclude that activation of the PLCγ/PKC pathway including PKC-α and PKC-ε is indispensable for Gab1 tyrosinephosphorylation and Gab1 plays a pivotal role in ERK activation in VSMC by PDGF.by guest on March 24, 2020 http://www.jbc.org/Downloaded from

Figure 1 :
Figure 1: Gab1 is tyrosine-phosphorylated by PDGF as well as EGF.

Figure 6 :
Figure 6: Effect of general tyrosine kinase inhibitors on PDGF-induced Gab1 tyrosine-

Figure 7 :
Figure 7: Effect of Gab1 overexpression and tyrosine-phosphorylation on ERK activity.