Stem cell factor induces phosphatidylinositol 3′-kinase–dependent Lyn/Tec/Dok-1 complex formation in hematopoietic cells

Blood ◽  
2000 ◽  
Vol 96 (10) ◽  
pp. 3406-3413 ◽  
Author(s):  
Thamar B. van Dijk ◽  
Emile van den Akker ◽  
Martine Parren-van Amelsvoort ◽  
Hiroyuki Mano ◽  
Bob Löwenberg ◽  
...  
Keyword(s):  
Stem Cell ◽  
Tyrosine Kinase ◽  
Stem Cell Factor ◽  
Hematopoietic Cells ◽  
Sh2 Domain ◽  
Stable Complex ◽  
Phosphatidylinositol 3 Kinase ◽  
Sh2 Domains ◽  
And Migration ◽  
Phosphatidylinositol 3

Stem cell factor (SCF) has an important role in the proliferation, differentiation, survival, and migration of hematopoietic cells. SCF exerts its effects by binding to cKit, a receptor with intrinsic tyrosine kinase activity. Activation of phosphatidylinositol 3′-kinase (PI3-K) by cKit was previously shown to contribute to many SCF-induced cellular responses. Therefore, PI3-K-dependent signaling pathways activated by SCF were investigated. The PI3-K-dependent activation and phosphorylation of the tyrosine kinase Tec and the adapter molecule p62Dok-1 are reported. The study shows that Tec and Dok-1 form a stable complex with Lyn and 2 unidentified phosphoproteins of 56 and 140 kd. Both the Tec homology and the SH2 domain of Tec were identified as being required for the interaction with Dok-1, whereas 2 domains in Dok-1 appeared to mediate the association with Tec. In addition, Tec and Lyn were shown to phosphorylate Dok-1, whereas phosphorylated Dok-1 was demonstrated to bind to the SH2 domains of several signaling molecules activated by SCF, including Abl, CrkL, SHIP, and PLCγ-1, but not those of Vav and Shc. These findings suggest that p62Dok-1 may function as an important scaffold molecule in cKit-mediated signaling.

Stem cell factor induces phosphatidylinositol 3′-kinase–dependent Lyn/Tec/Dok-1 complex formation in hematopoietic cells

Blood ◽  
2000 ◽  
Vol 96 (10) ◽  
pp. 3406-3413 ◽  
Author(s):  
Thamar B. van Dijk ◽  
Emile van den Akker ◽  
Martine Parren-van Amelsvoort ◽  
Hiroyuki Mano ◽  
Bob Löwenberg ◽  
...  
Keyword(s):  
Stem Cell ◽  
Tyrosine Kinase ◽  
Stem Cell Factor ◽  
Hematopoietic Cells ◽  
Sh2 Domain ◽  
Stable Complex ◽  
Phosphatidylinositol 3 Kinase ◽  
Sh2 Domains ◽  
And Migration ◽  
Phosphatidylinositol 3

Abstract Stem cell factor (SCF) has an important role in the proliferation, differentiation, survival, and migration of hematopoietic cells. SCF exerts its effects by binding to cKit, a receptor with intrinsic tyrosine kinase activity. Activation of phosphatidylinositol 3′-kinase (PI3-K) by cKit was previously shown to contribute to many SCF-induced cellular responses. Therefore, PI3-K-dependent signaling pathways activated by SCF were investigated. The PI3-K-dependent activation and phosphorylation of the tyrosine kinase Tec and the adapter molecule p62Dok-1 are reported. The study shows that Tec and Dok-1 form a stable complex with Lyn and 2 unidentified phosphoproteins of 56 and 140 kd. Both the Tec homology and the SH2 domain of Tec were identified as being required for the interaction with Dok-1, whereas 2 domains in Dok-1 appeared to mediate the association with Tec. In addition, Tec and Lyn were shown to phosphorylate Dok-1, whereas phosphorylated Dok-1 was demonstrated to bind to the SH2 domains of several signaling molecules activated by SCF, including Abl, CrkL, SHIP, and PLCγ-1, but not those of Vav and Shc. These findings suggest that p62Dok-1 may function as an important scaffold molecule in cKit-mediated signaling.

Phosphatidylinositol 3-kinase activation is mediated by high-affinity interactions between distinct domains within the p110 and p85 subunits

1994 ◽  
Vol 14 (1) ◽  
pp. 42-49
Author(s):  
K H Holt ◽  
L Olson ◽  
W S Moye-Rowley ◽  
J E Pessin
Keyword(s):  
Gene Fusion ◽  
Kinase Activity ◽  
Expression System ◽  
Sh2 Domain ◽  
Full Length ◽  
Transactivation Domain ◽  
Phosphatidylinositol 3 Kinase ◽  
Sh2 Domains ◽  
Amino Terminal ◽  
Phosphatidylinositol 3

Domains of interaction between the p85 and p110 subunits of phosphatidylinositol 3-kinase (PI 3-kinase) were studied with the yeast two-hybrid expression system. A gene fusion between the GAL4 transactivation domain and p85 activated transcription from a GAL1-lacZ reporter gene when complemented with a gene fusion between the GAL4 DNA binding domain and p110. To define subdomains responsible for this interaction, a series of p85 deletion mutants were analyzed. A 192-amino-acid inter-SH2 (IS) fragment (residues 429 to 621) was the smallest determinant identified that specifically associated with p110. In analogous experiments, the subdomain within p110 responsible for interaction with p85 was localized to an EcoRI fragment encoding the amino-terminal 127 residues. Expression of these two subdomains [p85(IS) with p110RI] resulted in 100-fold greater reporter activity than that obtained with full-length p85 and p110. Although the p85(IS) domain conferred a strong interaction with the p110 catalytic subunit, this region was not sufficient to impart phosphotyrosine peptide stimulation of PI 3-kinase activity. In contrast, coexpression of the p110 subunit with full-length p85 or with constructs containing the IS sequences flanked by both SH2 domains of p85 [p85(n/cSH2)] or either of the individual SH2 domains [p85(nSH2+IS) or p85(IS+cSH2)] resulted in PI 3-kinase activity that was activated by a phosphotyrosine peptide. These data suggest that phosphotyrosine peptide binding to either SH2 domain generates an intramolecular signal propagated through the IS region to allosterically activate p110.

scholarly journals Genetic analysis of a phosphatidylinositol 3-kinase SH2 domain reveals determinants of specificity.

1994 ◽  
Vol 14 (9) ◽  
pp. 5929-5938 ◽  
Author(s):  
M Yoakim ◽  
W Hou ◽  
Z Songyang ◽  
Y Liu ◽  
L Cantley ◽  
...  
Keyword(s):  
Phosphorylation Site ◽  
Growth Factor Receptor ◽  
Sh2 Domain ◽  
T Antigen ◽  
Site Directed Mutagenesis ◽  
Phosphatidylinositol 3 Kinase ◽  
Sh2 Domains ◽  
Src Homology ◽  
Polyomavirus Middle T Antigen ◽  
Phosphatidylinositol 3

Phosphatidylinositol 3-kinase is an important element in both normal and oncogenic signal transduction. Polyomavirus middle T antigen transforms cells in a manner depending on association of its tyrosine 315 phosphorylation site with Src homology 2 (SH2) domains on the p85 subunit of the phosphatidylinositol 3-kinase. Both nonselective and site-directed mutagenesis have been used to probe the interaction of middle T with the N-terminal SH2 domain of p85. Most of the 24 mutants obtained showed reduced middle T binding. However, mutations that showed increased binding were also found. Comparison of middle T binding to that of the platelet-derived growth factor receptor showed that some mutations altered the specificity of recognition by the SH2 domain. Mutations altering S-393, D-394, and P-395 were shown to affect the ability of the SH2 domain to select peptides from a degenerate phosphopeptide library. These results focus attention on the role of the EF loop in the SH2 domain in determining binding selectivity at the third position after the phosphotyrosine.

scholarly journals Phosphatidylinositol 3-kinase activation is mediated by high-affinity interactions between distinct domains within the p110 and p85 subunits.

1994 ◽  
Vol 14 (1) ◽  
pp. 42-49 ◽  
Author(s):  
K H Holt ◽  
L Olson ◽  
W S Moye-Rowley ◽  
J E Pessin
Keyword(s):  
Gene Fusion ◽  
Kinase Activity ◽  
Expression System ◽  
Sh2 Domain ◽  
Full Length ◽  
Transactivation Domain ◽  
Phosphatidylinositol 3 Kinase ◽  
Sh2 Domains ◽  
Amino Terminal ◽  
Phosphatidylinositol 3

Domains of interaction between the p85 and p110 subunits of phosphatidylinositol 3-kinase (PI 3-kinase) were studied with the yeast two-hybrid expression system. A gene fusion between the GAL4 transactivation domain and p85 activated transcription from a GAL1-lacZ reporter gene when complemented with a gene fusion between the GAL4 DNA binding domain and p110. To define subdomains responsible for this interaction, a series of p85 deletion mutants were analyzed. A 192-amino-acid inter-SH2 (IS) fragment (residues 429 to 621) was the smallest determinant identified that specifically associated with p110. In analogous experiments, the subdomain within p110 responsible for interaction with p85 was localized to an EcoRI fragment encoding the amino-terminal 127 residues. Expression of these two subdomains [p85(IS) with p110RI] resulted in 100-fold greater reporter activity than that obtained with full-length p85 and p110. Although the p85(IS) domain conferred a strong interaction with the p110 catalytic subunit, this region was not sufficient to impart phosphotyrosine peptide stimulation of PI 3-kinase activity. In contrast, coexpression of the p110 subunit with full-length p85 or with constructs containing the IS sequences flanked by both SH2 domains of p85 [p85(n/cSH2)] or either of the individual SH2 domains [p85(nSH2+IS) or p85(IS+cSH2)] resulted in PI 3-kinase activity that was activated by a phosphotyrosine peptide. These data suggest that phosphotyrosine peptide binding to either SH2 domain generates an intramolecular signal propagated through the IS region to allosterically activate p110.

Kit/stem cell factor receptor-induced activation of phosphatidylinositol 3′-kinase is essential for male fertility

10.1038/72814 ◽  
2000 ◽  
Vol 24 (2) ◽  
pp. 157-162 ◽  
Author(s):  
Peter Blume-Jensen ◽  
Guoqiang Jiang ◽  
Robert Hyman ◽  
Kuo-Fen Lee ◽  
Stephen O'Gorman ◽  
...  
Keyword(s):  
Stem Cell ◽  
Male Fertility ◽  
Stem Cell Factor ◽  
Phosphatidylinositol 3 Kinase ◽  
Phosphatidylinositol 3

scholarly journals The structure and function of p55PIK reveal a new regulatory subunit for phosphatidylinositol 3-kinase.

1995 ◽  
Vol 15 (8) ◽  
pp. 4453-4465 ◽  
Author(s):  
S Pons ◽  
T Asano ◽  
E Glasheen ◽  
M Miralpeix ◽  
Y Zhang ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Regulatory Subunit ◽  
Stable Complex ◽  
Phosphatidylinositol 3 Kinase ◽  
Sh2 Domains ◽  
Cellular Processes ◽  
Src Homology 2 ◽  
Src Homology ◽  
And Function ◽  
Phosphatidylinositol 3

Phosphatidylinositol 3-kinase (PI-3 kinase) is implicated in the regulation of diverse cellular processes, including insulin-stimulated glucose transport. PI-3 kinase is composed of a 110-kDa catalytic subunit and an 85-kDa regulatory subunit. Here, we describe p55PIK, a new regulatory subunit that was isolated by screening expression libraries with tyrosine-phosphorylated insulin receptor substrate 1 (IRS-1). p55PIK is composed of a unique 30-residue NH2 terminus followed by a proline-rich motif and two Src homology 2 (SH2) domains with significant sequence identify to those in p85. p55PIK mRNA is expressed early during development, remains abundant in adult mouse brain and testis tissue, and is detectable in adult adipocytes and heart and kidney tissues. p55PIK forms a stable complex with p110, and it associates with IRS-1 during insulin stimulation. Moreover, the activated insulin receptor phosphorylates p55PIK in Sf9 cells, and insulin stimulates p55PIK phosphorylation in CHOIR/p55PIK cells. The unique features of p55PIK suggest that it is important in receptor signaling.

scholarly journals Genetic analysis of a phosphatidylinositol 3-kinase SH2 domain reveals determinants of specificity

1994 ◽  
Vol 14 (9) ◽  
pp. 5929-5938
Author(s):  
M Yoakim ◽  
W Hou ◽  
Z Songyang ◽  
Y Liu ◽  
L Cantley ◽  
...  
Keyword(s):  
Phosphorylation Site ◽  
Growth Factor Receptor ◽  
Sh2 Domain ◽  
T Antigen ◽  
Site Directed Mutagenesis ◽  
Phosphatidylinositol 3 Kinase ◽  
Sh2 Domains ◽  
Src Homology ◽  
Polyomavirus Middle T Antigen ◽  
Phosphatidylinositol 3

Phosphatidylinositol 3-kinase is an important element in both normal and oncogenic signal transduction. Polyomavirus middle T antigen transforms cells in a manner depending on association of its tyrosine 315 phosphorylation site with Src homology 2 (SH2) domains on the p85 subunit of the phosphatidylinositol 3-kinase. Both nonselective and site-directed mutagenesis have been used to probe the interaction of middle T with the N-terminal SH2 domain of p85. Most of the 24 mutants obtained showed reduced middle T binding. However, mutations that showed increased binding were also found. Comparison of middle T binding to that of the platelet-derived growth factor receptor showed that some mutations altered the specificity of recognition by the SH2 domain. Mutations altering S-393, D-394, and P-395 were shown to affect the ability of the SH2 domain to select peptides from a degenerate phosphopeptide library. These results focus attention on the role of the EF loop in the SH2 domain in determining binding selectivity at the third position after the phosphotyrosine.

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

1993 ◽  
Vol 13 (3) ◽  
pp. 1737-1745
Author(s):  
J A Cooper ◽  
A Kashishian
Keyword(s):  
Fusion Protein ◽  
Fusion Proteins ◽  
Expression System ◽  
Sh2 Domain ◽  
Phosphorylation Sites ◽  
Phosphatidylinositol 3 Kinase ◽  
Binding Properties ◽  
Sh2 Domains ◽  
Phosphatidylinositol 3

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)

Physical and functional interactions between SH2 and SH3 domains of the Src family protein tyrosine kinase p59fyn

1994 ◽  
Vol 14 (9) ◽  
pp. 6372-6385
Author(s):  
G Panchamoorthy ◽  
T Fukazawa ◽  
L Stolz ◽  
G Payne ◽  
K Reedquist ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Enzymatic Activity ◽  
Sh3 Domain ◽  
Sh2 Domain ◽  
Phosphatidylinositol 3 Kinase ◽  
Biochemical Basis ◽  
Sh3 Domains ◽  
Protein Tyrosine ◽  
Family Protein ◽  
Phosphatidylinositol 3

The Src family protein tyrosine kinases participate in signalling through cell surface receptors that lack intrinsic tyrosine kinase domains. All nine members of this family possess adjacent Src homology (SH2 and SH3) domains, both of which are essential for repression of the enzymatic activity. The repression is mediated by binding between the SH2 domain and a C-terminal phosphotyrosine, and the SH3 domain is required for this interaction. However, the biochemical basis of functional SH2-SH3 interaction is unclear. Here, we demonstrate that when the SH2 and SH3 domains of p59fyn (Fyn) were present as adjacent domains in a single protein, binding of phosphotyrosyl peptides and proteins to the SH2 domain was enhanced, whereas binding of a subset of cellular polypeptide ligands to the SH3 domain was decreased. An interdomain communication was further revealed by occupancy with domain-specific peptide ligands: occupancy of the SH3 domain with a proline-rich peptide enhanced phosphotyrosine binding to the linked SH2 domain, and occupancy of the SH2 domain with phosphotyrosyl peptides enhanced binding of certain SH3-specific cellular polypeptides. Second, we demonstrate a direct binding between purified SH2 and SH3 domains of Fyn and Lck Src family kinases. Heterologous binding between SH2 and SH3 domains of closely related members of the Src family, namely, Fyn, Lck, and Src, was also observed. In contrast, Grb2, Crk, Abl, p85 phosphatidylinositol 3-kinase, and GTPase-activating protein SH2 domains showed lower or no binding to Fyn or Lck SH3 domains. SH2-SH3 binding did not require an intact phosphotyrosine binding pocket on the SH2 domain; however, perturbations of the SH2 domain induced by specific high-affinity phosphotyrosyl peptide binding abrogated binding of the SH3 domain. SH3-SH2 binding was observed in the presence of proline-rich peptides or when a point mutation (W119K) was introduced in the putative ligand-binding pouch of the Fyn SH3 domain, although these treatments completely abolished the binding to p85 phosphatidylinositol 3-kinase and other SH3-specific polypeptides. These biochemical SH2-SH3 interactions suggest novel mechanisms of regulating the enzymatic activity of Src kinases and their interactions with other proteins.

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