In vivo binding properties of SH2 domains from GTPase-activating protein and phosphatidylinositol 3-kinase.

We have used a transient expression system and mutant platelet-derived growth factor (PDGF) receptors to study the binding specificities of the Src homology 2 (SH2) regions of the Ras GTPase-activator protein (GAP) and the p85 alpha subunit of phosphatidylinositol 3-kinase (PI3 kinase). A number of fusion proteins, each tagged with an epitope allowing recognition by a monoclonal antibody, were expressed at levels comparable to those of endogenous GAP. Fusion proteins containing the central SH2-SH3-SH2 region of GAP or the C-terminal region of p85 alpha, which includes two SH2 domains, bound to PDGF receptors in response to PDGF stimulation. Both fusion proteins showed the same requirements for tyrosine phosphorylation sites in the PDGF receptor as the full-length proteins from which they were derived, i.e., binding of the GAP fusion protein was reduced by mutation of Tyr-771, and binding of the p85 fusion protein was reduced by mutation of Tyr-740, Tyr-751, or both residues. Fusion proteins containing single SH2 domains from either GAP or p85 alpha did not bind detectably to PDGF receptors in this system, suggesting that two SH2 domains in a single polypeptide cooperate to raise the affinity of binding. The sequence specificities of individual SH2 domains were deduced from the binding properties of fusion proteins containing one SH2 domain from GAP and another from p85. The results suggest that the C-terminal GAP SH2 domain specifies binding to Tyr-771, the C-terminal p85 alpha SH2 domain binds to either Tyr-740 or Tyr-751, and each protein's N-terminal SH2 domain binds to unidentified phosphorylation sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nck adapters are involved in the formation of dorsal ruffles, cell migration, and Rho signaling downstream of the platelet-derived growth factor beta receptor

The SH3 and SH2 domain-containing adapter proteins Nck1 and Nck2 are known to function downstream of activated tyrosine kinase receptors, such as the platelet-derived growth factor (PDGF) receptors. The SH2 domain of Nck1 binds to phosphorylated tyrosine residue 751 in PDGFbeta receptor and has been suggested to have a role in the PDGF-induced mobilization of the actin filament system. Because Tyr-751 is a site for additional receptor interactors, it has been difficult to discriminate the signaling from Nck from signaling via other molecules. For this reason we have used mouse embryonic fibroblasts derived from mice in which the genes for Nck1 and Nck2 have been inactivated by gene targeting (knock-out (KO) cells). The mutant cells had a reduced ability to form edge ruffles in response to PDGF, and the presence of Nck was obligatory for the formation of dorsal ruffles. In addition, the KO cells had a reduced chemotactic and migratory potential. Importantly, KO cells had reduced cell attachment properties and a reduced ability to form focal adhesions in response to serum stimulation. Moreover, signaling involving the Rho GTPases was defective in KO cells. In summary, our observations suggest that the Nck adapters are needed for signaling to Rho GTPases and actin dynamics downstream of the PDGFbeta receptor.     

Nckbeta adapter regulates actin polymerization in NIH 3T3 fibroblasts in response to platelet-derived growth factor bb

The SH3-SH3-SH3-SH2 adapter Nck represents a two-gene family that includes Nckalpha (Nck) and Nckbeta (Grb4/Nck2), and it links receptor tyrosine kinases to intracellular signaling networks. The function of these mammalian Nck genes has not been established. We report here a specific role for Nckbeta in platelet-derived growth factor (PDGF)-induced actin polymerization in NIH 3T3 cells. Overexpression of Nckbeta but not Nckalpha blocks PDGF-stimulated membrane ruffling and formation of lamellipoda. Mutation in either the SH2 or the middle SH3 domain of Nckbeta abolishes its interfering effect. Nckbeta binds at Tyr-1009 in human PDGF receptor beta (PDGFR-beta) which is different from Nckalpha's binding site, Tyr-751, and does not compete with phosphatidylinositol-3 kinase for binding to PDGFR. Microinjection of an anti-Nckbeta but not an anti-Nckalpha antibody inhibits PDGF-stimulated actin polymerization. Constitutively membrane-bound Nckbeta but not Nckalpha blocks Rac1-L62-induced membrane ruffling and formation of lamellipodia, suggesting that Nckbeta acts in parallel to or downstream of Rac1. This is the first report of Nckbeta's role in receptor tyrosine kinase signaling to the actin cytoskeleton.     

Interactions between SH2 domains and tyrosine-phosphorylated platelet-derived growth factor beta-receptor sequences: analysis of kinetic parameters by a novel biosensor-based approach.

The interaction between SH2 domains and phosphotyrosine-containing sequences was examined by real-time measurements of kinetic parameters. The SH2 domains of the p85 subunit of the phosphatidylinositol 3-kinase as well as of other signaling molecules were expressed in bacteria as glutathione S-transferase fusion proteins. Phosphotyrosine-containing peptides, corresponding to two autophosphorylation sites on the human platelet-derived growth factor beta-receptor that are responsible for phosphatidylinositol 3-kinase binding, were synthesized and used as capturing molecules, immobilized on a biosensor surface. The association and dissociation rate constants for binding to both sites were determined for intact p85 and the recombinant SH2 domains. High association rates were found to be coupled to very fast dissociation rates for all interactions studied. A binding specificity was observed for the two SH2 domains of p85, with the N-terminal SH2 binding with high affinity to the Tyr-751 site but not to the Tyr-740 site, and the C-terminal SH2 interacting strongly with both sites. This approach should be generally applicable to the study of the specificity inherent in the assembly of signaling complexes by activated protein-tyrosine kinase receptors.     

Molecular Dynamics Simulations Reveal that Tyr-317 Phosphorylation Reduces Shc Binding Affinity for Phosphotyrosyl Residues of Epidermal Growth Factor Receptor

The Src homology 2 (SH2) and collagen domain protein Shc plays a pivotal role in signaling via tyrosine kinase receptors, including epidermal growth factor receptor (EGFR). Shc binding to phospho-tyrosine residues on activated receptors is mediated by the SH2 and phospho-tyrosine binding (PTB) domains. Subsequent phosphorylation on Tyr-317 within the Shc linker region induces Shc interactions with Grb2-Son of Sevenless that initiate Ras-mitogen-activated protein kinase signaling. We use molecular dynamics simulations of full-length Shc to examine how Tyr-317 phosphorylation controls Shc conformation and interactions with EGFR. Our simulations reveal that Shc tyrosine phosphorylation results in a significant rearrangement of the relative position of its domains, suggesting a key conformational change. Importantly, computational estimations of binding affinities show that EGFR-derived phosphotyrosyl peptides bind with significantly more strength to unphosphorylated than to phosphorylated Shc. Our results unveil what we believe is a novel structural phenomenon, i.e., tyrosine phosphorylation of Shc within its linker region regulates the binding affinity of SH2 and PTB domains for phosphorylated Shc partners, with important implications for signaling dynamics.     

Modification of the 85-kilodalton subunit of phosphatidylinositol-3 kinase in platelet-derived growth factor-stimulated cells.

The activated platelet-derived growth factor (PDGF) receptor physically associates with p85, a subunit of phosphatidylinositol-3 kinase. Although this interaction may activate phosphatidylinositol-kinase and is crucial for PDGF-induced mitogenesis, it has not been shown whether p85 is modified in the process. p85 contains two SH2 (Src homology) domains, designated SH2-N and SH2-C. Recent experiments have shown that the SH2-C domain alone determines high-affinity binding of p85 to the PDGF receptor. The function of SH2-N, which binds receptors with lower affinity, is unknown. In this study, using a receptor-blotting technique, we find that p85 is modified by PDGF stimulation of intact cells. This modification involves inhibition of binding of the SH2-N region of p85 to the PDGF receptor. Studies with vanadate suggest that tyrosine phosphorylation of p85 is responsible for the modification of p85 detected by receptor blotting. Furthermore, recombinant p85 is modified in a similar manner when it is tyrosine phosphorylated in vitro by PDGF receptors. Tyrosine phosphorylation of p85 does not block binding of the SH2-C domain and therefore does not release p85 from high-affinity binding sites on the receptor in vitro. Instead, phosphorylation may regulate the ability of the SH2-N of p85 to bind to a different portion of the PDGF receptor or to another molecule in the signaling complex. This study provides the first evidence that p85 is tyrosine phosphorylated upon PDGF stimulation of cells and suggests that tyrosine phosphorylation of p85 regulates its activity or its interaction with other proteins.     

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.     

The SH2 and SH3 domain-containing Nck protein is oncogenic and a common target for phosphorylation by different surface receptors.

Signalling proteins such as phospholipase C-gamma (PLC-gamma) or GTPase-activating protein (GAP) of ras contain conserved regions of approximately 100 amino acids termed src homology 2 (SH2) domains. SH2 domains were shown to be responsible for mediating association between signalling proteins and tyrosine-phosphorylated proteins, including growth factor receptors. Nck is an ubiquitously expressed protein consisting exclusively of one SH2 and three SH3 domains. Here we show that epidermal growth factor or platelet-derived growth factor stimulation of intact human or murine cells leads to phosphorylation of Nck protein on tyrosine, serine, and threonine residues. Similar stimulation of Nck phosphorylation was detected upon activation of rat basophilic leukemia RBL-2H3 cells by cross-linking of the high-affinity immunoglobulin E receptors (Fc epsilon RI). Ligand-activated, tyrosine-autophosphorylated platelet-derived growth factor or epidermal growth factor receptors were coimmunoprecipitated with anti-Nck antibodies, and the association with either receptor molecule was mediated by the SH2 domain of Nck. Addition of phorbol ester was also able to stimulate Nck phosphorylation on serine residues. However, growth factor-induced serine/threonine phosphorylation of Nck was not mediated by protein kinase C. Interestingly, approximately fivefold overexpression of Nck in NIH 3T3 cells resulted in formation of oncogenic foci. These results show that Nck is an oncogenic protein and a common target for the action of different surface receptors. Nck probably functions as an adaptor protein which links surface receptors with tyrosine kinase activity to downstream signalling pathways involved in the control of cell proliferation.     

SH2 domains of the p85 alpha subunit of phosphatidylinositol 3-kinase regulate binding to growth factor receptors.

The binding of cytoplasmic signaling proteins such as phospholipase C-gamma 1 and Ras GTPase-activating protein to autophosphorylated growth factor receptors is directed by their noncatalytic Src homology region 2 (SH2) domains. The p85 alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase, which associates with several receptor protein-tyrosine kinases, also contains two SH2 domains. Both p85 alpha SH2 domains, when expressed individually as fusion proteins in bacteria, bound stably to the activated beta receptor for platelet-derived growth factor (PDGF). Complex formation required PDGF stimulation and was dependent on receptor tyrosine kinase activity. The bacterial p85 alpha SH2 domains recognized activated beta PDGF receptor which had been immobilized on a filter, indicating that SH2 domains contact autophosphorylated receptors directly. Several receptor tyrosine kinases within the PDGF receptor subfamily, including the colony-stimulating factor 1 receptor and the Steel factor receptor (Kit), also associate with PI 3-kinase in vivo. Bacterially expressed SH2 domains derived from the p85 alpha subunit of PI 3-kinase bound in vitro to the activated colony-stimulating factor 1 receptor and to Kit. We infer that the SH2 domains of p85 alpha bind to high-affinity sites on these receptors, whose creation is dependent on receptor autophosphorylation. The SH2 domains of p85 are therefore primarily responsible for the binding of PI 3-kinase to activated growth factor receptors.     

YXXL motifs in SH2-Bbeta are phosphorylated by JAK2, JAK1, and platelet-derived growth factor receptor and are required for membrane ruffling

SH2-Bbeta binds to the activated form of JAK2 and various receptor tyrosine kinases. It is a potent stimulator of JAK2, is required for growth hormone (GH)-induced membrane ruffling, and increases mitogenesis stimulated by platelet-derived growth factor (PDGF) and insulin-like growth factor I. Its domain structure suggests that SH2-Bbeta may act as an adapter protein to recruit downstream signaling proteins to kinase.SH2-Bbeta complexes. SH2-Bbeta is tyrosyl-phosphorylated in response to GH and interferon-gamma, stimulators of JAK2, as well as in response to PDGF and nerve growth factor. To begin to elucidate the role of tyrosyl phosphorylation in the function of SH2-Bbeta, we used phosphopeptide mapping, mutagenesis, and a phosphotyrosine-specific antibody to identify Tyr-439 and Tyr-494 in SH2-Bbeta as targets of JAK2 both in vitro and in intact cells. SH2-Bbeta lacking Tyr-439 and Tyr-494 inhibits GH-induced membrane ruffling but still activates JAK2. We provide evidence that JAK1, like JAK2, phosphorylates Tyr-439 and Tyr-494 in SH2-Bbeta and that PDGF receptor phosphorylates SH2-Bbeta on Tyr-439. Therefore, phosphorylated Tyr-439 and/or Tyr-494 in SH2-Bbeta may provide a binding site for one or more proteins linking cytokine receptor.JAK2 complexes and/or receptor tyrosine kinases to the actin cytoskeleton.