SH2 domains exhibit high-affinity binding to tyrosine-phosphorylated peptides yet also exhibit rapid dissociation and exchange.

src homology 2 (SH2) domains of intracellular signaling molecules such as phospholipase C-gamma and phosphatidylinositol 3'-kinase-associated protein p85 represent recognition motifs for specific phosphotyrosine-containing regions on activated growth factor receptors. The binding of SH2 domains to activated growth factor receptors controls the interaction with signaling molecules and the regulation of their activities. In this report, we describe the kinetic parameters and binding affinities of SH2 domains of p85 toward short phosphotyrosine-containing peptides with the amino acid sequence motif YMXM, derived from a major insulin receptor substrate, IRS-1, by using real time biospecific interaction analysis (BIAcore). Associations were specific and of very high affinity, with dissociation constants of 0.3 to 3 nM, between phosphopeptides and the two separate SH2 domains contained within p85. Nonphosphorylated peptides showed no measurable binding, and the interactions were specific for the primary sequence very close to the phosphotyrosine residue. Moreover, the interactions between phosphopeptides and SH2 domains of other signaling molecules were of much lower affinity. Interestingly, the binding of the SH2 domains to the tyrosine-phosphorylated peptides was of high affinity as a result of a very high on rate, of 3 x 10(7) to 40 x 10(7)/M/s; at the same time, the rate of dissociation, of 0.11 to 0.19/s, was rapid, allowing for rapid exchange of associating proteins with the tyrosine phosphorylation sites.

Download Full-text

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

Molecular and Cellular Biology ◽

10.1128/mcb.13.11.6889-6896.1993 ◽

1993 ◽

Vol 13 (11) ◽

pp. 6889-6896

Author(s):

R Nishimura ◽

W Li ◽

A Kashishian ◽

A Mondino ◽

M Zhou ◽

...

Keyword(s):

Growth Factor ◽

Binding Site ◽

Specific Binding ◽

Growth Factor Receptors ◽

Signaling Molecules ◽

Sh2 Domain ◽

Platelet Derived Growth Factor ◽

Pdgf Receptor ◽

Intact Cells ◽

Sh2 Domains

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.

Download Full-text

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

Molecular and Cellular Biology ◽

10.1128/mcb.13.11.6889 ◽

1993 ◽

Vol 13 (11) ◽

pp. 6889-6896 ◽

Cited By ~ 118

Author(s):

R Nishimura ◽

W Li ◽

A Kashishian ◽

A Mondino ◽

M Zhou ◽

...

Keyword(s):

Growth Factor ◽

Binding Site ◽

Specific Binding ◽

Growth Factor Receptors ◽

Signaling Molecules ◽

Sh2 Domain ◽

Platelet Derived Growth Factor ◽

Pdgf Receptor ◽

Intact Cells ◽

Sh2 Domains

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.

Download Full-text

Platelet-derived growth factor receptors form a high affinity state in membrane preparations. Kinetics and affinity cross-linking studies.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)42987-5 ◽

1984 ◽

Vol 259 (8) ◽

pp. 5287-5294 ◽

Cited By ~ 20

Author(s):

L T Williams ◽

P M Tremble ◽

M F Lavin ◽

M E Sunday

Keyword(s):

Growth Factor ◽

Growth Factor Receptors ◽

Platelet Derived Growth Factor ◽

Cross Linking ◽

High Affinity

Download Full-text

A new function for a phosphotyrosine phosphatase: linking GRB2-Sos to a receptor tyrosine kinase

Molecular and Cellular Biology ◽

10.1128/mcb.14.1.509-517.1994 ◽

1994 ◽

Vol 14 (1) ◽

pp. 509-517

Author(s):

W Li ◽

R Nishimura ◽

A Kashishian ◽

A G Batzer ◽

W J Kim ◽

...

Keyword(s):

Growth Factor ◽

Intracellular Signaling ◽

Egf Receptor ◽

Tyrosine Phosphatase ◽

Growth Factor Receptors ◽

Ras Signaling ◽

Pdgf Receptor ◽

Phosphotyrosine Phosphatase ◽

Src Homology

Autophosphorylated growth factor receptors provide binding sites for the src homology 2 domains of intracellular signaling molecules. In response to epidermal growth factor (EGF), the activated EGF receptor binds to a complex containing the signaling protein GRB2 and the Ras guanine nucleotide-releasing factor Sos, leading to activation of the Ras signaling pathway. We have investigated whether the platelet-derived growth factor (PDGF) receptor binds GRB2-Sos. In contrast with the EGF receptor, the GRB2 does not bind to the PDGF receptor directly. Instead, PDGF stimulation induces the formation of a complex containing GRB2; 70-, 80-, and 110-kDa tyrosine-phosphorylated proteins; and the PDGF receptor. Moreover, GRB2 binds directly to the 70-kDa protein but not to the PDGF receptor. Using a panel of PDGF beta-receptor mutants with altered tyrosine phosphorylation sites, we identified Tyr-1009 in the PDGF receptor as required for GRB2 binding. Binding is inhibited by a phosphopeptide containing a YXNX motif. The protein tyrosine phosphatase Syp/PTP1D/SHPTP2/PTP2C is approximately 70 kDa, binds to the PDGF receptor via Tyr-1009, and contains several YXNX sequences. We found that the 70-kDa protein that binds to the PDGF receptor and to GRB2 comigrates with Syp and is recognized by anti-Syp antibodies. Furthermore, both GRB2 and Sos coimmunoprecipitate with Syp from lysates of PDGF-stimulated cells, and GRB2 binds directly to tyrosine-phosphorylated Syp in vitro. These results indicate that GRB2 interacts with different growth factor receptors by different mechanisms and the cytoplasmic phosphotyrosine phosphatase Syp acts as an adapter between the PDGF receptor and the GRB2-Sos complex.

Download Full-text

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

Molecular and Cellular Biology ◽

10.1128/mcb.12.3.991-997.1992 ◽

1992 ◽

Vol 12 (3) ◽

pp. 991-997

Author(s):

C J McGlade ◽

C Ellis ◽

M Reedijk ◽

D Anderson ◽

G Mbamalu ◽

...

Keyword(s):

Growth Factor ◽

Tyrosine Kinases ◽

Growth Factor Receptors ◽

Alpha Subunit ◽

Receptor Protein ◽

Regulatory Subunit ◽

Pdgf Receptor ◽

Colony Stimulating Factor ◽

Sh2 Domains ◽

Stimulating Factor

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.

Download Full-text