Phosphospecific antibodies reveal temporal regulation of platelet-derived growth factor beta receptor signaling

The platelet-derived growth factor beta receptor (betaPDGFR) is a receptor tyrosine kinase involved in multiple aspects of cell growth and differentiation. Upon activation, betaPDGFR is phosphorylated at up to nine different tyrosine residues. Phosphorylation of the receptor results in at least two different outcomes: recruitment of signaling molecules and activation of intrinsic receptor kinase activity. In order to evaluate the phosphorylation state of the receptor, phosphospecific antibodies were generated against peptides encompassing betaPDGFR phospho-Y751, phospho-Y771, or phospho-Y857. When phosphorylated, these sites enable the receptor to recruit signaling molecules PI3K or RasGAP, or enhance the receptor's kinase activity, respectively. We found that receptors phosphorylated at Y751, Y771, and Y857 display distinct temporal and spatial distribution by immunofluorescence. Subsequent biochemical studies revealed that receptor function corresponding to each of the phosphorylated sites was regulated as a function of time. Within the first 10 min, PDGF enhanced the receptor's kinase activity and initiated recruitment of PI3K and RasGAP. After prolonged exposure to PDGF, PI3K binding persisted to approximately 85% of the amount bound at 10 min, whereas binding of RasGAP and the exogenous kinase activity of the receptor diminished to less than 15% of the levels displayed at 10 min. We conclude that the phosphorylation state of the receptor, as well as its signaling capacity, is dynamic and changes as cells are continuously exposed to PDGF.     

GTPase-activating protein and phosphatidylinositol 3-kinase bind to distinct regions of the platelet-derived growth factor receptor beta subunit.

In response to binding of platelet-derived growth factor (PDGF), the PDGF receptor (PDGFR) beta subunit is phosphorylated on tyrosine residues and associates with numerous signal transduction enzymes, including the GTPase-activating protein of ras (GAP) and phosphatidylinositol 3-kinase (PI3K). Previous studies have shown that association of PI3K requires phosphorylation of tyrosine 751 (Y751) in the kinase insert and that this region of receptor forms at least a portion of the binding site for PI3K. In this study, the in vitro binding of GAP to the PDGFR was investigated. Like PI3K, GAP associates only with receptors that have been permitted to autophosphorylate, and GAP itself does not require tyrosine phosphate in order to stably associate with the phosphorylated PDGFR. To define which tyrosine residues are required for GAP binding, a panel of PDGFR phosphorylation site mutants was tested. Mutation of Y771 reduced the amount of GAP that associates to an undetectable level. In contrast, the F771 (phenylalanine at 771) mutant bound wild-type levels of PI3K, whereas the F740 and F751 mutants bound 3 and 23%, respectively, of the wild-type levels of PI3K but wild-type levels of GAP. The F740/F751 double mutant associated with wild-type levels of GAP, but no detectable PI3K activity, while the F740/F751/F771 triple mutant could not bind either GAP or PI3K. The in vitro and in vivo associations of GAP and PI3K activity to these PDGFR mutants were indistinguishable. The distinct tyrosine residue requirements suggest that GAP and PI3K bind different regions of the PDGFR. This possibility was also supported by the observation that the antibody to the PDGFR kinase insert Y751 region that blocks association of PI3K had only a minor effect on the in vitro binding of GAP. In addition, highly purified PI3K and GAP associated in the absence of other cellular proteins and neither cooperated nor competed with each other's binding to the PDGFR. Taken together, these studies indicate that GAP and PI3K bind directly to the PDGFR and have discrete binding sites that include portions of the kinase insert domain.     

Phosphorylation of the PDGF receptor beta subunit creates a tight binding site for phosphatidylinositol 3 kinase.

The beta subunit of the platelet derived growth factor receptor (PDGFR) coprecipitates with a phosphatidyl-inositol 3 kinase activity (PI3K) following stimulation of cells by PDGF. Mutagenesis of a tyrosine (Y) phosphorylation site, Y751, in the PDGFR, greatly reduces PI3K, consistent with the possibility that phosphorylation of Y751 signals association of PI3K. To test this we have reconstituted the binding of the PDGFR beta subunit and PI3K in vitro. Binding is rapid, saturable and requires phosphorylation of the PDGFR at Y751, but does not require PDGF-dependent phosphorylation of PI3K. To test which portions of the PDGFR are important for binding, we used an antibody to a small region of the receptor that includes Y751. This antibody blocked in vitro binding of PI3K to the receptor, while an antiserum to the C-terminus of the receptor had no effect on binding of PI3K. In addition, we found that PDGF stimulation of a cell results in the association of essentially all the PI3K activity with cellular PDGFRs. These data suggest that PI3K is a specific ligand for PDGF receptors that are phosphorylated at Y751.     

Mutation of tyrosine residue 857 in the PDGF β-receptor affects cell proliferation but not migration

Activation of platelet-derived growth factor (PDGF) receptors occurs through ligand-induced dimerization and autophosphorylation. In this study, we investigated the effects of mutation of tyrosine residue 857 (Y857) in the activation loop of the PDGF β-receptor (PDGFRβ) to phenylalanine (Y857F). In agreement with previous observations, we found that PDGFRβ^Y857F had a severely diminished in vitro kinase activity. However, in vivo the overall amount of tyrosine phosphorylation of PDGFRβ^Y857F was similar to that of the wild-type receptor, except for the tyrosine residue 771 (Y771) which displayed a stronger phosphorylation in the mutant receptor. Analysis of the ability to induce signal transduction revealed that the PDGFRβ^Y857F mutant had an attenuated activation of Akt and Erk1/2 MAP kinase. In contrast, the mutant receptor efficiently mediated phosphorylation of the ubiquitin-ligase c-Cbl that participates in receptor internalization and degradation, and PLCγ which has previously been shown to be connected with various cellular responses, including migration. However, the protein tyrosine phosphatase SHP-2, implicated in the PDGF-induced mitogenic response, together with the adaptor proteins Alix and Stam, involved in intracellular sorting of receptor, was not phosphorylated in cells expressing PDGFRβ^Y857F. We found that both receptor variants were internalized from the cell surface and degraded at a comparable rate. Interestingly, PDGFRβ^Y857F was unable to mediate PDGF-BB-induced mitogenic signaling, whereas it could elicit a chemotactic response.     

A-Raf associates with and regulates platelet-derived growth factor receptor signalling

Raf kinases are important intermediates in epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) mediated activation of the mitogen-activated protein kinase (MAPK) pathway. In this report, we show that the A-Raf kinase is associated with activated EGF receptor complexes and with PDGF receptor (PDGFR) complexes independent of prior PDGF treatment. The ability of A-Raf to associate with receptor tyrosine kinases could provide a Ras-GTP-independent mechanism for the membrane localization of A-Raf. Expression of a partially activated A-Raf mutant resulted in decreased tyrosine phosphorylation of the PDGFR, specifically on Y857 (autophosphorylation site) and Y1021 (phospholipase Cgamma1 (PLCgamma1) binding site), but not the binding sites for other signalling proteins (Nck, phosphatidylinositol 3'-kinase (PI3K), RasGAP, Grb2, SHP). Activated A-Raf expression also altered the activation of PLCgamma1, and p85-associated PI3K. Thus, A-Raf can regulate PLCgamma1 signalling via a PDGFR-dependent mechanism and may also regulate PI3K signalling via a PDGFR-independent mechanism.     

Tyrosines 1021 and 1009 are phosphorylation sites in the carboxy terminus of the platelet-derived growth factor receptor beta subunit and are required for binding of phospholipase C gamma and a 64-kilodalton protein, respectively.

Binding of platelet-derived growth factor (PDGF) to the PDGF receptor (PDGFR) beta subunit triggers receptor tyrosine phosphorylation and the stable association of a number of signal transduction molecules, including phospholipase C gamma (PLC gamma), the GTPase activating protein of ras (GAP), and phosphatidylinositol-3 kinase (PI3K). Previous reports have identified three PDGFR tyrosine phosphorylation sites in the kinase insert domain that are important for stable association of GAP and PI3K. Two of them, tyrosine (Y) 740, and Y-751 are required for the stable association of PI3K, while Y-771 is required for binding of GAP. Here we present data for two additional tyrosine phosphorylation sites, Y-1009 and Y-1021, that are both in the carboxy-terminal region of the PDGFR. Characterization of PDGFR mutants in which these phosphorylation sites are substituted with phenylalanine (F) indicated that Y-1021 and Y-1009 were required for the stable association of PLC gamma and a 64-kDa protein, respectively. An F-1009/F-1021 double mutant selectively failed to bind both PLC gamma and the 64-kDa protein, whereas all of the carboxy-terminal mutants bound wild-type levels of GAP and PI3K. The carboxy terminus encodes the complete binding site for PLC gamma, since a phosphorylated carboxy-terminal fusion protein selectively bound PLC gamma. To determine the biological consequences of failure to associate with PLC gamma, we measured PDGF-dependent inositol phosphate production and initiation of DNA synthesis. The PDGFR mutants that failed to associate with PLC gamma were not able to mediate the PDGF-dependent production of inositol phosphates. Since tyrosine phosphorylation of PLC gamma enhances its enzymatic activity, we speculated that PDGFR mutants that failed to activate PLC gamma were unable to mediate its tyrosine phosphorylation. Surprisingly, the F-1021 receptor mediated readily detectable levels of PDGF-dependent PLC gamma tyrosine phosphorylation. Thus, the production of inositol phosphates requires not only PLC gamma tyrosine phosphorylation but also its association with the PDGFR. Comparison of the mutant PDGFRs' abilities to initiate PDGF-dependent DNA synthesis indicated that failure to associate with PLC gamma and produce inositol phosphates diminished the mitogenic response by 30%. In contrast, preventing the PDGFR from binding the 64-kDa protein did not compromise PDGF-triggered DNA synthesis at saturating concentrations of PDGF. Thus, it appears that phosphorylation of the PDGFR at Y-1021 is required for the stable association of PLC gamma to the receptor's carboxy terminus, the production of inositol phosphates, and initiation of the maximal mitogenic response.     

Phosphorylation sites in the PDGF receptor with different specificities for binding GAP and PI3 kinase in vivo.

Tyrosine residues have been identified in the human platelet-derived growth factor (PDGF) receptor beta-subunit whose phosphorylation is stimulated by PDGF. These sites are also in vitro autophosphorylation sites. There are a total of three phosphorylation sites in the kinase insert region, tyrosines 740, 751 and 771. Mutagenesis studies show that Tyr740 and 751 are involved in the PDGF-stimulated binding of phosphatidylinositol (PI) 3 kinase, and Tyr771 is required for efficient binding of GAP, the GTPase activator of Ras. The requirement for Tyr751 is only detected at low PDGF receptor levels, suggesting that it increases the affinity of binding of PI3 kinase but is not absolutely required. Small deletions in the kinase insert only 10 residues from Tyr740 and Tyr771 do not significantly reduce binding of PI3 kinase or GAP, indicating that distant sequences are probably unimportant for recognition. The data suggest that the receptor signals to different pathways via different phosphorylated tyrosines, and that certain proteins, such as PI3 kinase, can recognize two phosphorylated tyrosines in a single receptor.     

Platelet-derived growth factor-dependent activation of phosphatidylinositol 3-kinase is regulated by receptor binding of SH2-domain-containing proteins which influence Ras activity.

Upon binding of platelet-derived growth factor (PDGF), the PDGF beta receptor (PDGFR) undergoes autophosphorylation on distinct tyrosine residues and binds several SH2-domain-containing signal relay enzymes, including phosphatidylinositol 3-kinase (PI3K), phospholipase C gamma (PLC gamma), the GTPase-activating protein of Ras (RasGAP), and the tyrosine phosphatase SHP-2. In this study, we have investigated whether PDGF-dependent PI3K activation is affected by the other proteins that associate with the PDGFR. We constructed and characterized a series of PDGFR mutants which contain binding sites for PI3K as well as one additional protein, either RasGAP, SHP-2, or PLC gamma. While all of the receptors had wild-type levels of PDGF-stimulated tyrosine kinase activity and associated with comparable amounts of PI3K activity, their abilities to trigger accumulation of PI3K products in vivo differed dramatically. The wild-type receptor, as well as receptors that recruited PI3K or PI3K and SHP-2, were all capable of fully activating PI3K. In contrast, receptors that associated with PI3K and RasGAP or PI3K and PLC gamma displayed a greatly reduced ability to stimulate production of PI3K products. When this series of receptors was tested for their ability to activate Ras, we observed a strong positive correlation between Ras activation and PI3K activation. Further investigation of the relationship between Ras and PI3K indicated that Ras was upstream of PI3K. Thus, activation of PI3K requires not only binding of PI3K to the tyrosine-phosphorylated PDGFR but accumulation of GTP-bound Ras as well. Furthermore, PLC gamma and RasGAP negatively modulate PDGF-dependent PI3K activation. Finally, PDGF-stimulated signal relay can be regulated by altering the ratio of SH2-domain-containing enzymes that are recruited to the PDGFR.     

Functions of the major tyrosine phosphorylation site of the PDGF receptor beta subunit.

Two tyrosine phosphorylation sites in the human platelet-derived growth factor receptor (PDGFR) beta subunit have been mapped previously to tyrosine (Y)751, in the kinase insert, and Y857, in the kinase domain. Y857 is the major site of tyrosine phosphorylation in PDGF-stimulated cells. To evaluate the importance of these phosphorylations, we have characterized the wild-type (WT) and mutant human PDGF receptor beta subunits in dog kidney epithelial cells. Replacement of either Y751 or Y857 with phenylalanine (F) reduced PDGF-stimulated DNA synthesis to approximately 50% of the WT level. A mutant receptor with both tyrosines mutated was unable to initiate DNA synthesis, as was a kinase-inactive mutant receptor. Transmodulation of the epidermal growth factor receptor required Y857 but not Y751. We also tested the effects of phosphorylation site mutations on PDGF-stimulated receptor kinase activity. PDGF-induced tyrosine phosphorylation of two cellular proteins, phospholipase C gamma 1 (PLC gamma 1) and the GTPase activating protein of Ras (GAP), was assayed in epithelial cells expressing each of the mutant receptors. Tyrosine phosphorylation of GAP and PLC gamma 1 was reduced markedly by the F857 mutation but not significantly by the F751 mutation. Reduced kinase activity of F857 receptors was also evident in vitro. Immunoprecipitated WT receptors showed a two- to fourfold increase in specific kinase activity if immunoprecipitated from PDGF-stimulated cells. The F751 receptors showed a similar increase in activity, but F857 receptors did not. Our data suggest that phosphorylation of Y857 may be important for stimulation of kinase activity of the receptors and for downstream actions such as epidermal growth factor receptor transmodulation and mitogenesis.     

Constitutive diffuse activation of phosphoinositide 3-kinase at the plasma membrane by v-Src suppresses the chemotactic response to PDGF by abrogating the polarity of PDGF receptor signalling

Cancer cells depend on chemotaxis for invasion and frequently overexpress and/or activate Src. We previously reported that v-Src accelerates motility by promoting phosphoinositide 3-kinase (PI3-K) signalling but abrogates chemotaxis. We here addressed the mechanism of the loss of chemotactic response to platelet-derived growth factor (PDGF) gradients in fibroblasts harbouring a thermosensitive v-Src kinase. At non-permissive temperature, PDGF receptor (PDGFR) signalling, assessed by phosphoY(751)-specific antibodies (a docking site for PI3-K), was not detected without PDGF and showed a concentration-dependent PDGF response. Both immunolabeling of PI3-K (p110) and live cell imaging of its product (phosphatidylinositol 3,4,5 tris-phosphate) showed PI3-K recruitment and activation at lamellipodia polarized towards a PDGF gradient. Centrosomes and PDGFR- and Src-bearing endosomes were also oriented towards this gradient. Upon v-Src thermoactivation, (i) Y(751) phosphorylation was moderately induced without PDGF and synergistically increased with PDGF; (ii) PI3-K was recruited and activated all along the plasma membrane without PDGF and did not polarize in response to a PDGF gradient; and (iii) polarization of centrosomes and of PDGFR-bearing endosomes were also abrogated. Thus, PDGF can further increase PDGFR auto-phosphorylation despite strong Src kinase activity, but diffuse downstream activation of PI3-K by Src abrogates cell polarization and chemotaxis: "signalling requires silence".     

PDGF-mediated PI3K/AKT/β-catenin signaling regulates gap junctions in corpus cavernosum smooth muscle cells

Erectile dysfunction (ED) is the most common sexual disorder that men report to healthcare providers. Gap junctions (GJs) are thought to be responsible for synchronous shrinkage of corpus cavernosum smooth muscle cells (CCSMCs), and play thus an important role in the maintenance of an erection. Hypoxia has been suggested as a pathological mechanism underlying ED. Here we demonstrate that hypoxia increased the expression of platelet-derived growth factor (PDGF) and the main GJ component connexin (Cx)43 in CCSMCs. Inhibiting PDGF receptor (PDGFR) activity decreased Cx43 expression. Treatment with different concentrations of PDGF increased the levels of phosphorylated protein kinase B (AKT), β-catenin, and Cx43, whereas inhibition of PDGFR or activation of phosphatidylinositol 3 kinase (PI3K)/AKT signaling altered β-catenin and Cx43 expression. Meanwhile, silencing β-catenin resulted in the downregulation of Cx43. These results demonstrate that PDGF secretion by CCSMCs and vascular endothelial cells is enhanced under hypoxic conditions, leading to increased Cx43 expression through PI3K/AKT/β-catenin signaling and ultimately affecting GJ function in ED. Thus, targeting this pathway is a potential therapeutic strategy for the treatment of ED.     

Two signaling molecules share a phosphotyrosine-containing binding site in the platelet-derived growth factor receptor.

Autophosphorylation sites of growth factor receptors with tyrosine kinase activity function as specific binding sites for Src homology 2 (SH2) domains of signaling molecules. This interaction appears to be a crucial step in a mechanism by which receptor tyrosine kinases relay signals to downstream signaling pathways. Nck is a widely expressed protein consisting exclusively of SH2 and SH3 domains, the overexpression of which causes cell transformation. It has been shown that various growth factors stimulate the phosphorylation of Nck and its association with autophosphorylated growth factor receptors. A panel of platelet-derived growth factor (PDGF) receptor mutations at tyrosine residues has been used to identify the Nck binding site. Here we show that mutation at Tyr-751 of the PDGF beta-receptor eliminates Nck binding both in vitro and in living cells. Moreover, the Y751F PDGF receptor mutant failed to mediate PDGF-stimulated phosphorylation of Nck in intact cells. A phosphorylated Tyr-751 is also required for binding of phosphatidylinositol-3 kinase to the PDGF receptor. Hence, the SH2 domains of p85 and Nck share a binding site in the PDGF receptor. Competition experiments with different phosphopeptides derived from the PDGF receptor suggest that binding of Nck and p85 is influenced by different residues around Tyr-751. Thus, a single tyrosine autophosphorylation site is able to link the PDGF receptor to two distinct SH2 domain-containing signaling molecules.     

Autophosphorylation of the PDGF receptor in the kinase insert region regulates interactions with cell proteins

We have identified two platelet-derived growth factor (PDGF)-dependent autophosphorylation sites in the beta subunit of the human PDGF receptor (PDGF-R). The major site of phosphorylation (Tyr-857) corresponds to the major autophosphorylation site in many other tyrosine kinases. Tyr-751, which lies within the kinase insert region, is a second in vivo site and the major in vitro site. Immunoprecipitates of wild-type PDGF-Rs prepared from PDGF-treated cells contained a phosphatidylinositol (PI) 3 kinase activity and three specific polypeptides as well as the PDGF-R. Mutation of Tyr-751 to Phe or Gly, or mutation of the catalytic domain to abolish kinase activity, blocked association of the PDGF-R with the PI kinase and the three proteins. These results suggest that autophosphorylation in the kinase insert region triggers the binding of the activated PDGF-R to specific cellular proteins, including a PI kinase whose activity is known to be stimulated by PDGF. Thus autophosphorylation may play a novel role in signal transduction via the PDGF-R.     

Receptor association and tyrosine phosphorylation of S6 kinases

Ribosomal protein S6 kinase (S6K) is activated by an array of mitogenic stimuli and is a key player in the regulation of cell growth. The activation process of S6 kinase involves a complex and sequential series of multiple Ser/Thr phosphorylations and is mainly mediated via phosphatidylinositol 3-kinase (PI3K)-3-phosphoinositide-dependent protein kinase-1 (PDK1) and mTor-dependent pathways. Upstream regulators of S6K, such as PDK1 and protein kinase B (PKB/Akt), are recruited to the membrane via their pleckstrin homology (PH) or protein-protein interaction domains. However, the mechanism of integration of S6K into a multi-enzyme complex around activated receptor tyrosine kinases is not clear. In the present study, we describe a specific interaction between S6K with receptor tyrosine kinases, such as platelet-derived growth factor receptor (PDGFR). The interaction with PDGFR is mediated via the kinase or the kinase extension domain of S6K. Complex formation is inducible by growth factors and leads to S6K tyrosine phosphorylation. Using PDGFR mutants, we have shown that the phosphorylation is exerted via a PDGFR-src pathway. Furthermore, src kinase phosphorylates and coimmunoprecipitates with S6K in vivo. Inhibitors towards tyrosine kinases, such as genistein and PP1, or src-specific SU6656, but not PI3K and mTor inhibitors, lead to a reduction in tyrosine phosphorylation of S6K. In addition, we mapped the sites of tyrosine phosphorylation in S6K1 and S6K2 to Y39 and Y45, respectively. Mutational and immunofluorescent analysis indicated that phosphorylation of S6Ks at these sites does not affect their activity or subcellular localization. Our data indicate that S6 kinase is recruited into a complex with RTKs and src and becomes phosphorylated on tyrosine/s in response to PDGF or serum.     

The GTPase-activating protein of Ras suppresses platelet-derived growth factor beta receptor signaling by silencing phospholipase C-gamma 1.

The beta receptor for platelet-derived growth factor (beta PDGFR) is activated by binding of PDGF and undergoes phosphorylation at multiple tyrosine residues. The tyrosine-phosphorylated receptor associates with numerous SH2-domain-containing proteins which include phospholipase C-gamma 1 (PLC gamma), the GTPase-activating protein of Ras (GAP), the p85 subunit of phosphatidylinositol 3 kinase (PI3K), the phosphotyrosine phosphatase Syp, and several other proteins. Our previous studies indicated that PI3K and PLC gamma were required for relay of the mitogenic signal of beta PDGFR, whereas GAP and Syp did not appear to be required for this response. In this study, we further investigated the role of GAP and Syp in mitogenic signaling by beta PDGFR. Focusing on the PLC gamma-dependent branch of beta PDGFR signaling, we constructed a series of mutant beta PDGFRs that contained the binding sites for pairs of the receptor-associated proteins: PLC gamma and PI3K, PLC gamma and GAP, or PLC gamma and Syp. Characterization of these mutants showed that while all receptors were catalytically active and bound similar amounts of PLC gamma, they differed dramatically in their ability to initiate DNA synthesis. This signaling deficiency related to an inability to efficiently tyrosine phosphorylate and activate PLC gamma. Surprisingly, the crippled receptor was the one that recruited PLC gamma and GAP. Thus, GAP functions to suppress signal relay by the beta PDGFR, and it does so by silencing PLC gamma. These findings demonstrate that the biological response to PDGF depends not only on the ability of the beta PDGFR to recruit signal relay enzymes but also on the blend of these receptor-associated proteins.     

Phosphorylation of PKCδ by FER tips the balance from EGFR degradation to recycling

Receptor degradation terminates signaling by activated receptor tyrosine kinases. Degradation of EGFR occurs in lysosomes and requires the switching of RAB5 for RAB7 on late endosomes to enable their fusion with the lysosome, but what controls this critical switching is poorly understood. We show that the tyrosine kinase FER alters PKCδ function by phosphorylating it on Y374, and that phospho-Y374-PKCδ prevents RAB5 release from nascent late endosomes, thereby inhibiting EGFR degradation and promoting the recycling of endosomal EGFR to the cell surface. The rapid association of phospho-Y374-PKCδ with EGFR-containing endosomes is diminished by PTPN14, which dephosphorylates phospho-Y374-PKCδ. In triple-negative breast cancer cells, the FER-dependent phosphorylation of PKCδ enhances EGFR signaling and promotes anchorage-independent cell growth. Importantly, increased Y374-PKCδ phosphorylation correlating with arrested late endosome maturation was identified in ∼25% of triple-negative breast cancer patients, suggesting that dysregulation of this pathway may contribute to their pathology.     

A phosphatidylinositol-3 kinase binds to platelet-derived growth factor receptors through a specific receptor sequence containing phosphotyrosine.

Platelet-derived growth factor (PDGF) stimulates autophosphorylation of the PDGF receptor and association of the receptor with several cytoplasmic molecules, including phosphatidylinositol-3 kinase (PI3 kinase). In this study we examined the association of PI3 kinase with immunoprecipitated autophosphorylated PDGF receptor in vitro. The PI3 kinase from cell lysates bound to the wild-type receptor but not to a mutant receptor that had a deletion of the kinase insert region. A protein of an apparent size of 85 kDa bound to the receptor, consistent with previous observations that a protein of this size is associated with PI3 kinase activity. In addition, 110- and 74-kDa proteins bound to the phosphorylated receptor. Dephosphorylated receptors lost the ability to bind PI3 kinase activity as well as the 85-kDa protein. A 20-amino-acid peptide composed of a sequence in the kinase insert region that included one of the autophosphorylation sites of the receptor (tyrosine 719) as well as a nearby tyrosine (Y708) blocked the binding of PI3 kinase to the receptor, but only when the peptide was phosphorylated on tyrosine residues. A scrambled version of the peptide did not block PI3 kinase binding to the receptor even when it was phosphorylated on tyrosine. These tyrosine-phosphorylated peptides did not block binding of phospholipase C-gamma or GTPase-activating protein to the receptor. In separate experiments (receptor blots), soluble radiolabeled receptor bound specifically to an 85-kDa protein present in sodium dodecyl sulfate-polyacrylamide gel electrophoresis-fractionated 3T3 cell lysates that were transferred to nitrocellulose paper. The binding was blocked by the same tyrosine-phosphorylated peptides that prevented binding of PI3 kinase activity to immobilized receptors. These findings show that the PDGF receptor binds directly to an 85-kDa protein and to a PI3 kinase activity through specific sequences in the kinase insert region. The association of a 110-kDa protein with the receptor also involve these sequences, suggesting that this protein may be a subunit of the PI3 kinase. Phosphotyrosine is an essential structure required for the interactions of these proteins with the PDGF receptor.     

Catalytically inactive SHIP2 inhibits proliferation by attenuating PDGF signaling in 3T3-L1 preadipocytes

Inadequate proliferation and/or differentiation of preadipocytes may lead to adipose tissue dysfunction characterized by hypertrophied, insulin-resistant adipocytes. Platelet-derived growth factor (PDGF) may alter adipose tissue function by promoting proliferation of preadipocytes. Two principal signaling pathways that regulate proliferation are PI3K/PI(3,4,5)P3/Akt and Shc/Ras/ERK1/2. SH2 domain-containing inositol 5-phosphatase 2 (SHIP2) dephosphorylates PI(3,4,5)P3, and also binds to Shc. Our goal was to determine how SHIP2 affects these PDGF signaling routes. To assess the role of the 5-phosphatase domain, we expressed wild-type or catalytically inactive dominant-negative SHIP2 (P686A-D690A-R691A; PDR/AAA) in 3T3-L1 preadipocytes. Surprisingly, SHIP2 PDR/AAA inhibited proliferation more potently than wild-type SHIP2. After three days of proliferation, phospho-Akt, phospho-ERK1/2, and PDGF receptor (PDGFR) levels were reduced in PDR/AAA-expressing preadipocytes. SHIP2 PDR/AAA interference with PDGFR signaling was demonstrated using imatinib, an inhibitor of PDGFR tyrosine kinase. The anti-proliferative effect of imatinib observed in control preadipocytes was not significant in SHIP2 PDR/AAA-expressing preadipocytes, indicating a pre-existing impairment of PDGFR-dependent mitogenesis in these cells. The inhibition of PDGF-activated mitogenic pathways by SHIP2 PDR/AAA was consistent with a decrease in PDGFR phosphorylation caused by a drop in receptor levels in SHIP2 PDR/AAA-expressing cells. SHIP2 PDR/AAA promoted ubiquitination of the PDGFR and its degradation via the lysosomal pathway independently of the association between the E3 ubiquitin ligase c-Cbl and PDGFR. Overall, our findings indicate that SHIP2 PDR/AAA reduces preadipocyte proliferation by attenuating PDGFR signaling.