Linker region of Akt1/protein kinase Balpha mediates platelet-derived growth factor-induced translocation and cell migration

The phosphatidylinositol 3-kinase (PI3K) signaling pathway(s) is activated by a variety of agonists to regulate cell migration. Here, we show that the stimulation of mouse embryonic fibroblasts with platelet-derived growth factor (PDGF) induces migration in a PI3K-dependent manner. Cells lacking Akt1/PKBalpha exhibit impaired migration and peripheral ruffling in response to PDGF stimulation, whereas cells lacking Akt2/PKBbeta are normal. In addition, over-expression of Akt1/PKBalpha but not Akt2/PKBbeta is sufficient to restore PDGF-induced cell migration in an Akt1/PKBalpha and Akt2/PKBbeta deficient background. In response to PDGF stimulation, Akt1/PKBalpha selectively translocates to membrane ruffles, however, this localization is abrogated by substituting the linker region of Akt2/PKBbeta. Similarly, expression of an Akt2/PKBalpha chimera containing the linker region of Akt1/PKBalpha restored PDGF-induced migration in cells lacking both Akt1/PKBalpha and Akt2/PKBbeta. Finally, over-expression of constitutively active Rac rescues PDGF-induced migration defects in cells lacking Akt1/PKBalpha. Given these results, we suggest that Akt1/PKBalpha controls cell migration by selectively translocating to the leading edge and activating Rac.     

Nox4 and Duox1/2 Mediate Redox Activation of Mesenchymal Cell Migration by PDGF

Platelet derived growth factor (PDGF) orchestrates wound healing and tissue regeneration by regulating recruitment of the precursor mesenchymal stromal cells (MSC) and fibroblasts. PDGF stimulates generation of hydrogen peroxide that is required for cell migration, but the sources and intracellular targets of H₂O₂ remain obscure. Here we demonstrate sustained live responses of H₂O₂ to PDGF and identify PKB/Akt, but not Erk1/2, as the target for redox regulation in cultured 3T3 fibroblasts and MSC. Apocynin, cell-permeable catalase and LY294002 inhibited PDGF-induced migration and mitotic activity of these cells indicating involvement of PI3-kinase pathway and H₂O₂. Real-time PCR revealed Nox4 and Duox1/2 as the potential sources of H₂O₂. Silencing of Duox1/2 in fibroblasts or Nox4 in MSC reduced PDGF-stimulated intracellular H₂O₂, PKB/Akt phosphorylation and migration, but had no such effect on Erk1/2. In contrast to PDGF, EGF failed to increase cytoplasmic H₂O₂, phosphorylation of PKB/Akt and migration of fibroblasts and MSC, confirming the critical impact of redox signaling. We conclude that PDGF-induced migration of mesenchymal cells requires Nox4 and Duox1/2 enzymes, which mediate redox-sensitive activation of PI3-kinase pathway and PKB/Akt.     

The role of focal adhesion kinase-phosphatidylinositol 3-kinase-akt signaling in hepatic stellate cell proliferation and type I collagen expression

Following a fibrogenic stimulus, the hepatic stellate cell (HSC) undergoes a complex activation process associated with increased cell proliferation and excess deposition of type I collagen. The focal adhesion kinase (FAK)-phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway is activated by platelet-derived growth factor (PDGF) in several cell types. We investigated the role of the FAK-PI3K-Akt pathway in HSC activation. Inhibition of FAK activity blocked HSC migration, cell attachment, and PDGF-induced PI3K and Akt activation. Both serum- and PDGF-induced Akt phosphorylation was inhibited by LY294002, an inhibitor of PI3K. A constitutively active form of Akt stimulated HSC proliferation in serum-starved HSCs, whereas LY294002 and dominant-negative forms of Akt and FAK inhibited PDGF-induced proliferation. Transforming growth factor-beta, an inhibitor of HSC proliferation, did not block PDGF-induced Akt phosphorylation, suggesting that transforming growth factor-beta mediates its antiproliferative effect downstream of Akt. Expression of type I collagen protein and alpha1(I) collagen mRNA was increased by Akt activation and inhibited when PI3K activity was blocked. Therefore, FAK is important for HSC migration, cell attachment, and PDGF-induced cell proliferation. PI3K is positioned downstream of FAK. Signals for HSC proliferation are transduced through FAK, PI3K, and Akt. Finally, expression of type I collagen is regulated by the PI3K-Akt signaling pathway.     

The S1P2 receptor negatively regulates platelet-derived growth factor-induced motility and proliferation

Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite, is the ligand for five specific G protein-coupled receptors, named S1P(1) to S1P(5). In this study, we found that cross-communication between platelet-derived growth factor receptor and S1P(2) serves as a negative damper of PDGF functions. Deletion of the S1P(2) receptor dramatically increased migration of mouse embryonic fibroblasts toward S1P, serum, and PDGF but not fibronectin. This enhanced migration was dependent on expression of S1P(1) and sphingosine kinase 1 (SphK1), the enzyme that produces S1P, as revealed by downregulation of their expression with antisense RNA and small interfering RNA, respectively. Although S1P(2) deletion had no significant effect on tyrosine phosphorylation of the PDGF receptors or activation of extracellular signal-regulated kinase 1/2 or Akt induced by PDGF, it reduced sustained PDGF-dependent p38 phosphorylation and markedly enhanced Rac activation. Surprisingly, S1P(2)-null cells not only exhibited enhanced proliferation but also markedly increased SphK1 expression and activity. Conversely, reintroduction of S1P(2) reduced DNA synthesis and expression of SphK1. Thus, S1P(2) serves as a negative regulator of PDGF-induced migration and proliferation as well as SphK1 expression. Our results suggest that a complex interplay between PDGFR and S1P receptors determines their functions.     

TGFbeta intercepts nuclear glycogen synthase kinase 3beta to inhibit PDGF-induced DNA synthesis in mesangial cells

Here, we demonstrate a mechanism of TGFbeta-mediated inhibition of PDGF-induced DNA synthesis in mesangial cells. TGFbeta significantly inhibited nuclear Akt phosphorylation without any effect on PDGF-stimulated phosphorylation of PDGFR at PI 3 kinase binding site (Tyr-751). Remarkably, TGFbeta inhibited cyclin D1 and cyclin E expression with concomitant decrease in CDK2 activity induced by PDGF. More importantly, we demonstrate that TGFbeta significantly abolished Akt-mediated serine-9 phosphorylation of glycogen synthase kinase 3beta (GSK3beta), thus prevented its inactivation. Expression of inactive GSK3betaK85R mutant increased cyclin D1 expression and DNA synthesis similar to PDGF. These results provide the first evidence that TGFbeta intercepts Akt kinase activity in the nucleus to block inactivation of GSK3beta, leading to attenuation of PDGF-induced CDK2 activity and DNA synthesis.     

NHERF2 increases platelet-derived growth factor-induced proliferation through PI-3-kinase/Akt-, ERK-, and Src family kinase-dependent pathway

Platelet-derived growth factor (PDGF) has multiple functions including inhibition of apoptosis and promotion of cell proliferation. In this study, we show that Na(+)/H(+) exchanger regulatory factor 2 (NHERF2) binds to the carboxyl-terminal PDZ domain-binding motif of the PDGF receptor through a PDZ domain-mediated interaction, and evaluate the consequence on PDGF-induced proliferation. Stable transfection with NHERF2 increased the PDGF-induced phosphorylation of ERK and Akt in Rat1 embryonic fibroblasts. The phosphorylation of Akt was blocked by pretreatment with LY294002, a PI-3-kinase inhibitor, in both Rat1/NHERF2 and Rat1/vector cells. In Rat1/vector cells, PDGF-induced phosphorylation of ERK was completely inhibited by pretreatment with PD98059, a MEK inhibitor. In contrast, the NHERF2-dependent increase of ERK phosphorylation was not affected by pretreatment with PD98059 in Rat1/NHERF2 cells. Thus, the NHERF2-dependent increase of ERK phosphorylation occurs in a MEK-independent fashion. Pretreatment with PP2, a specific inhibitor of Src family tyrosine kinase, completely blocked the NHERF2-dependent increase of the phosphorylation of ERK and Akt, suggesting that NHERF2 up-regulates Erk phosphorylation through a Src family kinase-dependent pathway. Consistent with these results, the PDGF-induced thymidine incorporation was increased in Rat1/NHERF2 cells, and the NHERF2-dependent increase of thymidine incorporation was prevented by treatment with LY294002 and PP2 but not with PD98059. These results suggest that NHERF2 stimulates PDGF-induced proliferation by increasing PI-3-kinase/Akt, MEKindependent ERK, and Src family kinase-mediated signaling pathways.     

PDGF-induced vascular smooth muscle cell proliferation is associated with dysregulation of insulin receptor substrates

In vascular smooth muscle cells (VSMCs), platelet-derived growth factor (PDGF) plays a major role in inducing phenotypic switching from contractile to proliferative state. Importantly, VSMC phenotypic switching is also determined by the phosphorylation state/expression levels of insulin receptor substrate (IRS), an intermediary signaling component that is shared by insulin and IGF-I. To date, the roles of PDGF-induced key proliferative signaling components including Akt, p70S6kinase, and ERK1/2 on the serine phosphorylation/expression of IRS-1 and IRS-2 isoforms remain unclear in VSMCs. We hypothesize that PDGF-induced VSMC proliferation is associated with dysregulation of insulin receptor substrates. Using human aortic VSMCs, we demonstrate that prolonged PDGF treatment led to sustained increases in the phosphorylation of protein kinases such as Akt, p70S6kinase, and ERK1/2, which mediate VSMC proliferation. In addition, PDGF enhanced IRS-1/IRS-2 serine phosphorylation and downregulated IRS-2 expression in a time- and concentration-dependent manner. Notably, phosphoinositide 3-kinase (PI 3-kinase) inhibitor (PI-103) and mammalian target of rapamycin inhibitor (rapamycin), which abolished PDGF-induced Akt and p70S6kinase phosphorylation, respectively, blocked PDGF-induced IRS-1 serine phosphorylation and IRS-2 downregulation. In contrast, MEK1/ERK inhibitor (U0126) failed to block PDGF-induced IRS-1 serine phosphorylation and IRS-2 downregulation. PDGF-induced IRS-2 downregulation was prevented by lactacystin, an inhibitor of proteasomal degradation. Functionally, PDGF-mediated IRS-1/IRS-2 dysregulation resulted in the attenuation of insulin-induced IRS-1/IRS-2-associated PI 3-kinase activity. Pharmacological inhibition of PDGF receptor tyrosine kinase with imatinib prevented IRS-1/IRS-2 dysregulation and restored insulin receptor signaling. In conclusion, strategies to inhibit PDGF receptors would not only inhibit neointimal growth but may provide new therapeutic options to prevent dysregulated insulin receptor signaling in VSMCs in nondiabetic and diabetic states.     

Cooperative regulation of GSK-3 by muscarinic and PDGF receptors is associated with airway myocyte proliferation

Muscarinic receptors and platelet-derived growth factor (PDGF) receptors synergistically induce proliferation of airway smooth muscle (ASM), but the pathways that regulate these effects are not yet completely identified. We hypothesized that glycogen synthase kinase-3 (GSK-3), a kinase that represses several promitogenic signaling pathways in its unphosphorylated form, is cooperatively inhibited by PDGF and muscarinic receptors in immortalized human ASM cell lines. PDGF or methacholine alone induced rapid GSK-3 phosphorylation. This phosphorylation was sustained only for PDGF; however, methacholine potentiated PDGF-induced sustained GSK-3 phosphorylation. Synergistic effects of methacholine also were observed on PDGF-induced retinoblastoma protein (Rb) phosphorylation and cell proliferation. Suppression of GSK-3 inhibitory function using SB 216763 also augmented PDGF-induced Rb phosphorylation and cell cycle progression; this synergy was similar in magnitude to that seen for methacholine with PDGF. GSK-3 phosphorylation induced by methacholine required PKC, since it was abolished by GF 109203X and G? 6976; however, inhibition of PKC had no effect on cell responses to PDGF. PKC inhibition also specifically abolished the synergistic effect of methacholine on PDGF-induced GSK-3 phosphorylation and cell proliferation. Collectively, these results show that GSK-3 plays a key repressive role in ASM cell proliferation. Moreover, muscarinic receptors mediate PKC-dependent GSK-3 inhibition, and this appears to be a primary mechanism underpinning augmentation of PDGF-induced cell growth.     

Inactivation of platelet-derived growth factor receptor by the tumor suppressor PTEN provides a novel mechanism of action of the phosphatase

PTEN, mutated in a variety of human cancers, is a dual specificity protein phosphatase and also possesses D3-phosphoinositide phosphatase activity on phosphatidylinositol 3,4,5-tris-phosphate (PIP(3)), a product of phosphatidylinositol 3-kinase. This PIP(3) phosphatase activity of PTEN contributes to its tumor suppressor function by inhibition of Akt kinase, a direct target of PIP(3). We have recently shown that Akt regulates PDGF-induced DNA synthesis in mesangial cells. In this study, we demonstrate that expression of PTEN in mesangial cells inhibits PDGF-induced Akt activation leading to reduction in PDGF-induced DNA synthesis. As a potential mechanism, we show that PTEN inhibits PDGF-induced protein tyrosine phosphorylation with concomitant dephosphorylation and inactivation of tyrosine phosphorylated and activated PDGF receptor. Recombinant as well as immunopurified PTEN dephosphorylates autophosphorylated PDGF receptor in vitro. Expression of phosphatase deficient mutant of PTEN does not dephosphorylate PDGF-induced tyrosine phosphorylated PDGF receptor. Rather its expression increases tyrosine phosphorylation of PDGF receptor. Furthermore, expression of PTEN attenuated PDGF-induced signal transduction including phosphatidylinositol 3-kinase and Erk1/2 MAPK activities. Our data provide the first evidence that PTEN is physically associated with platelet-derived growth factor (PDGF) receptor and that PDGF causes its dissociation from the receptor. Finally, we show that both the C2 and tail domains of PTEN contribute to binding to the PDGF receptor. These data demonstrate a novel aspect of PTEN function where it acts as an effector for the PDGF receptor function and negatively regulates PDGF receptor activation.     

Sponge-derived acetylenic alcohols,petrosiols, inhibit proliferation and migration of platelet-derived growth factor (PDGF)-induced vascular smooth muscle cells

Platelet-derived growth factor (PDGF) induces the proliferation and migration of vascular smooth muscle cells (VSMCs), leading to the development of various vascular disorders such as restenosis and atherosclerosis. Therefore, inhibitors of PDGF-induced cellular events would be candidate agents for treating these diseases. During the search for such inhibitors from marine sources, we isolated petrosiols A–D (1–4) and related compounds from the marine sponge Petrosia strongylata. These metabolites, which we previously reported as neurotrophic substances, showed an inhibitory effect on PDGF-induced DNA synthesis at IC₅₀ values of 0.69–2.2 μM. Petrosiol A (1) inhibited PDGF-induced cell proliferation without remarkable cytotoxicity and arrested cell cycle progression from the G0/G1 to S phase by inducing the downregulation of the expression of G1 checkpoint proteins cyclin D1, cyclin E, cyclin-dependent kinases (CDK)2, and CDK4 and the upregulation of the expression of p21 and p27. In addition, petrosiol A (1) inhibited the phosphorylation of PDGF receptor-β and its downstream proteins such as phospholipase C (PLC)-γ1, Akt, and extracellular signal-regulated kinase (ERK)1/2. These results suggest that 1 inhibited PDGF-induced VSMC proliferation by interrupting the phosphorylation of PDGF receptor-β followed by downstream signal transduction. Furthermore, petrosiol A (1) suppressed PDGF-induced actin filament dissociation and cell migration, suggesting that 1 and its derivatives may be used for the prevention and treatment of vascular diseases.     

Transforming Growth Factor β Inhibits Platelet Derived Growth Factor-Induced Vascular Smooth Muscle Cell Proliferation via Akt-Independent,Smad-Mediated Cyclin D1 Downregulation

In adult tissue, vascular smooth muscle cells (VSMCs) exist in a differentiated phenotype, which is defined by the expression of contractile proteins and lack of proliferation. After vascular injury, VSMC adopt a synthetic phenotype associated with proliferation, migration and matrix secretion. The transition between phenotypes is a consequence of the extracellular environment, and in particular, is regulated by agonists such as the pro-differentiating cytokine transforming growth factor β (TGFβ) and the pro-proliferative cytokine platelet derived growth factor (PDGF). In this study, we investigated the interplay between TGFβ and PDGF with respect to their ability to regulate VSMC proliferation. Stimulation of human aortic VSMC with TGFβ completely blocked proliferation induced by all isoforms of PDGF, as measured by DNA synthesis and total cell number. Mechanistically, PDGF-induced Cyclin D1 mRNA and protein expression was inhibited by TGFβ. TGFβ had no effect on PDGF activation of its receptor and ERK1/2, but inhibited Akt activation. However, constitutively active Akt did not reverse the inhibitory effect of TGFβ on Cyclin D1 expression even though inhibition of the proteasome blocked the effect of TGFβ. siRNA against Smad4 completely reversed the inhibitory effect of TGFβ on PDGF-induced Cyclin D1 expression and restored proliferation in response to PDGF. Moreover, siRNA against KLF5 prevented Cyclin D1 upregulation by PDGF and overexpression of KLF5 partially reversed TGFβ-induced inhibition of Cyclin D1 expression. Taken together, our results demonstrate that KLF5 is required for PDGF-induced Cyclin D1 expression, which is inhibited by TGFβ via a Smad dependent mechanism, resulting in arrest of VSMCs in the G1 phase of the cell cycle.     

Protein Disulfide Isomerase Is Required for Platelet-derived Growth Factor-induced Vascular Smooth Muscle Cell Migration, Nox1 NADPH Oxidase Expression, and RhoGTPase Activation

Vascular Smooth Muscle Cell (VSMC) migration into vessel neointima is a therapeutic target for atherosclerosis and postinjury restenosis. Nox1 NADPH oxidase-derived oxidants synergize with growth factors to support VSMC migration. We previously described the interaction between NADPH oxidases and the endoplasmic reticulum redox chaperone protein disulfide isomerase (PDI) in many cell types. However, physiological implications, as well as mechanisms of such association, are yet unclear. We show here that platelet-derived growth factor (PDGF) promoted subcellular redistribution of PDI concomitant to Nox1-dependent reactive oxygen species production and that siRNA-mediated PDI silencing inhibited such reactive oxygen species production, while nearly totally suppressing the increase in Nox1 expression, with no change in Nox4. Furthermore, PDI silencing inhibited PDGF-induced VSMC migration assessed by distinct methods, whereas PDI overexpression increased spontaneous basal VSMC migration. To address possible mechanisms of PDI effects, we searched for PDI interactome by systems biology analysis of physical protein-protein interaction networks, which indicated convergence with small GTPases and their regulator RhoGDI. PDI silencing decreased PDGF-induced Rac1 and RhoA activities, without changing their expression. PDI co-immunoprecipitated with RhoGDI at base line, whereas such association was decreased after PDGF. Also, PDI co-immunoprecipitated with Rac1 and RhoA in a PDGF-independent way and displayed detectable spots of perinuclear co-localization with Rac1 and RhoGDI. Moreover, PDI silencing promoted strong cytoskeletal changes: disorganization of stress fibers, decreased number of focal adhesions, and reduced number of RhoGDI-containing vesicular recycling adhesion structures. Overall, these data suggest that PDI is required to support Nox1/redox and GTPase-dependent VSMC migration.     

Involvement of Phosphatidylinositide 3′-Kinase and Rac in Platelet-Derived Growth Factor-Induced Actin Reorganization and Chemotaxis

Previous work has suggested a role for phosphatidylinositide 3′-kinase (PI3-kinase) in platelet-derived growth factor (PDGF)-induced actin reorganization and chemotaxis. In support of this notion, we show in this report that the PI3-kinase inhibitor wortmannin inhibits chemotaxis of PDGF β-receptor expressing porcine aortic endothelial (PAE/PDGFR-β) cells. Treatment with wortmannin resulted in a dose-dependent decrease in chemotaxis with an IC₅₀value of about 15–20 nM.Higher concentrations of wortmannin also reduced basal random migration of transfected cells in the absence of PDGF. We also investigated the role of Rac in PDGF-induced actin reorganization and cell motility. Overexpression of wt Rac in PAE/PDGFR-β cells led to an increased cell motility and edge ruffling in response to PDGF-BB, compared to control cells. In PAE/PDGFR-β cells transfected with inducible V12Rac (a constitutively active Rac mutant), membrane ruffling occurred in the absence of PDGF stimulation and was independent of PI3-kinase activity. On the other hand, PAE/PDGFR-β cells transfected with inducible N17Rac (a dominant negative Rac mutant) failed to show membrane ruffling in response to PDGF stimulation. Together with previous observations, these data indicate that activation of PI3-kinase is crucial for initiation of PDGF-induced cell motility responses and that Rac has a major role downstream of PI3-kinase, in this pathway.     

Regulation of Raf by Akt controls growth and differentiation in vascular smooth muscle cells.

The stimulation of platelet-derived growth factor (PDGF) receptors shifts vascular smooth muscle (VSM) cells toward a more proliferative phenotype. Thrombin activates the same signaling cascades in VSM cells, namely the Ras/Raf/MEK/ERK and the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt pathways. Nonetheless, thrombin was not mitogenic, but rather increased the expression of the smooth muscle-specific myosin heavy chain (SM-MHC) indicative of an in vitro re-differentiation of VSM cells. A more detailed analysis of the temporal pattern and relative signal intensities revealed marked differences. The strong and biphasic phosphorylation of ERK1/2 in response to thrombin correlated with its ability to increase the activity of the SM-MHC promoter whereas Akt was only partially and transiently phosphorylated. By contrast, PDGF, a potent mitogen in VSM cells, induced a short-lived ERK1/2 phosphorylation but a complete and sustained phosphorylation of Akt. The phosphorylated form of Akt physically interacted with Raf. Moreover, Akt phosphorylated Raf at Ser(259), resulting in a reduced Raf kinase activity and a termination of MEK and ERK1/2 phosphorylation. Disruption of the PI 3-kinase signaling prevented the PDGF-induced Akt and Raf-Ser(259) phosphorylation. Under these conditions, PDGF elicited a more sustained MEK and ERK phosphorylation and increased SM-MHC promoter activity. Consistently, in cells that express dominant negative Akt, PDGF increased SM-MHC promoter activity. Furthermore, expression of constitutively active Akt blocked the thrombin-stimulated SM-MHC promoter activity. Thus, we present evidence that the balance and cross-regulation between the PI 3-kinase/Akt and Ras/Raf/MEK signaling cascades determine the temporal pattern of ERK1/2 phosphorylation and may thereby guide the phenotypic modulation of vascular smooth muscle cells.     

Role of c-Jun N-terminal kinase in the PDGF-induced proliferation and migration of human adipose tissue-derived mesenchymal stem cells

Platelet-derived growth factor (PDGF) is a critical regulator of proliferation and migration for mesenchymal type cells. In this study, we examined the role of mitogen-activated protein (MAP) kinases in the PDGF-BB-induced proliferation and migration of human adipose tissue-derived mesenchymal stem cells (hATSCs). The PDGF-induced proliferation was prevented by a pretreatment with the c-Jun N-terminal kinase (JNK) inhibitor, SP600125. However, it was not prevented by a pretreatment with a p38 MAP kinase inhibitor, SB202190, and a specific inhibitor of the upstream kinase of extracellular signal-regulated kinase (ERK1/2), U0126. Treatment with PDGF induced the activation of JNK and ERK in hATSCs, and pretreatment with SP600125 specifically inhibited the PDGF-induced activation of JNK. Treatment with PDGF induced the cell cycle transition from the G0/G1 phase to the S phase, the elevated expression of cyclin D1, and the phosphorylation of Rb, which were prevented by a pretreatment with SP600125. In addition, the PDGF-induced migration of hATSCs was completely blocked by a pretreatment with SP600125, but not with U0126 and SB202190. These results suggest that JNK protein kinase plays a key role in the PDGF-induced proliferation and migration of mesenchymal stem cells.     

Tyr740 and Tyr751 residues of platelet-derived growth factor beta receptor are responsible for the redox regulation of phosphatase and tensin homolog in the cells stimulated with platelet-derived growth factor

Exposure of cells to hydrogen peroxide or platelet-derived growth factor (PDGF) induced Akt phosphorylation and oxidation of phosphatase and tensin homolog (PTEN). The Cys124 and Cys71 residues of PTEN were critical for the formation of a disulfide bond and the intermediate glutathionylation in the process of reduction of the disulfide bond. To determine which specific tyrosine residues of the PDGF beta receptor (PDGFβR) is involved in PDGF-induced PTEN oxidation and Akt phosphorylation, we investigated a kinase activity-deficient mutant and PDGFβR mutants where the tyrosine residues in the binding site for phosphoinositide 3-kinase (PI3K), GTPase-activating protein of Ras, Src homology 2 domain containing protein-tyrosine phosphatase-2, and phospholipase C-1 were replaced by Phe. Both PTEN oxidation and Akt phosphorylation did not occur in response to PDGF in the kinase-deficient mutant and in the PDGFβR mutant with a mutation in the PI3K binding site (Tyr740 and Tyr751). Thus, the kinase activity and the constituent Tyr740 and Tyr751 residues of PDGFβR in the cells stimulated with PDGF are responsible for the oxidation of PTEN and the Akt phosphorylation.     

The mitochondrial reactive oxygen species regulator p66Shc controls PDGF-induced signaling and migration through protein tyrosine phosphatase oxidation

Growth factor receptors induce a transient increase in reactive oxygen species (ROS) levels upon receptor binding to promote signaling through oxidation of protein tyrosine phosphatases (PTPs). Most studies have focused on NADPH oxidases as the dominant source of ROS to induce PTP oxidation. A potential additional regulator of growth factor-induced PTP oxidation is p66Shc, which stimulates mitochondrial ROS production. This study explores the contribution of p66Shc-induced ROS to PTP oxidation and growth factor receptor-induced signaling and migration through analyses of p66Shc-KO fibroblasts and cells with siRNA-mediated p66Shc downregulation. Analyses of PDGFβR phosphorylation in two independent cell systems demonstrated a decrease in PDGFβR phosphorylation after p66Shc deletion or downregulation, which occurred in a partially site-selective and antioxidant-sensitive manner. Deletion of p66Shc also reduced PDGF-induced activation of downstream signaling of Erk, Akt, PLCγ-1, and FAK. Importantly, reduced levels of p66Shc led to decreased oxidation of DEP1, PTP1B, and SHP2 after PDGF stimulation. The cell biological relevance of these findings was indicated by demonstration of a significantly reduced migratory response in PDGF-stimulated p66Shc-KO fibroblasts, consistent with reduced PDGFβR-Y1021 and PLCγ-1 phosphorylation. Downregulation of p66Shc also reduced EGFR phosphorylation and signaling, indicating that the positive role of p66Shc in receptor tyrosine kinase signaling is potentially general. Moreover, downregulation of the mitochondrial hydrogen peroxide scavenger peroxiredoxin 3 increased PDGFβR phosphorylation, showing that mitochondrial ROS in general promote PDGFβR signaling. This study thus identifies a previously unrecognized role for p66Shc in the regulation of PTP oxidation controlling growth factor-induced signaling and migration. In more general terms, the study indicates a regulatory role for mitochondrial-derived ROS in the control of PTP oxidation influencing growth factor signaling.