Intrinsic regulation of the interactions between the SH3 domain of p85 subunit of phosphatidylinositol-3 kinase and the protein network of BCR/ABL oncogenic tyrosine kinase

2005 ◽  
Vol 33 (10) ◽  
pp. 1222-1228 ◽  
Author(s):  
Shu-yue Ren ◽  
Fan Xue ◽  
Jan Feng ◽  
Tomasz Skorski
Keyword(s):  
Tyrosine Kinase ◽  
Sh3 Domain ◽  
Protein Network ◽  
Phosphatidylinositol 3 Kinase ◽  
Intrinsic Regulation ◽  
Phosphatidylinositol 3

Intrinsic Regulation of the Interactions between the SH3 Domain of p85 Subunit of Phosphatidylinositol-3 Kinase and BCR/ABL Oncogenic Tyrosine Kinase.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2961-2961
Author(s):  
Shuyue Ren ◽  
Fan Xue ◽  
Jan Feng ◽  
Tomasz Skorski
Keyword(s):  
Catalytic Activity ◽  
Tyrosine Kinase ◽  
Philadelphia Chromosome ◽  
Sh3 Domain ◽  
Myelogenous Leukemia ◽  
Wild Type ◽  
Phosphatidylinositol 3 Kinase ◽  
Abl Kinase ◽  
Intrinsic Regulation ◽  
Phosphatidylinositol 3

Abstract BCR/ABL fusion tyrosine kinase is responsible for the initiation and maintenance of the Philadelphia chromosome (Ph1)-positive chronic myelogenous leukemia (CML) and a cohort of acute lymphocytic leukemias (ALL). Our previous studies showed that a signaling protein phosphatidylinositol-3 kinase (PI-3k) is essential for the growth of CML cells, but not of normal hematopoietic cells, and that p85 subunit of PI-3k co-immunoprecipitates with BCR/ABL (Skorski et al., (1995) Blood 86, 726–36; Skorski et al., (1997) Embo J 16, 6151–61; Klejman et al., (2002) Oncogene 21, 5868–76). Therefore, we made an attempt to better characterize the p85 - BCR/ABL interactions. Here we show that SH3 domain of p85 (p85-SH3) pulls-down the p210BCR/ABL kinase from hematopoietic cell lysates. In addition, we characterize the p85-SH3 mutants, which abrogate or enhance this interaction. The results of pull-down assays of the p85-SH3 mutants seem to support the assumption that p85-SH3 interacts with the BCR/ABL protein network via the proline-rich (PxxP) region. One of the surprising findings was the enhanced binding affinity of the tyrosine to phenylalanine p85-SH3 domain mutants in comparison to the wild-type p85-SH3. Based on these results we speculate on the capability of p85-SH3 to interact with BCR/ABL and on the p85-SH3 conformational requirements necessary for this reaction. PxxP - binding appears to be required for the interaction of p85-SH3 with BCR/ABL protein complex and activation of the catalytic activity of PI-3k, whereas subsequent BCR/ABL-dependent phosphorylation of the tyrosines may facilitate the release of activated PI-3k from the complex.

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.

scholarly journals 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.

Structural studies of the phosphatidylinositol 3-kinase (PI3K) SH3 domain in complex with a peptide ligand: role of the anchor residue in ligand binding

2010 ◽  
Vol 391 (1) ◽  
Author(s):  
Renu Batra-Safferling ◽  
Joachim Granzin ◽  
Susanne Mödder ◽  
Silke Hoffmann ◽  
Dieter Willbold
Keyword(s):  
Crystal Structure ◽  
Ligand Binding ◽  
Protein Interactions ◽  
Intracellular Signaling ◽  
Sh3 Domain ◽  
Peptide Ligand ◽  
Type I ◽  
Phosphatidylinositol 3 Kinase ◽  
Anchor Residue ◽  
Phosphatidylinositol 3

Abstract Src homology 3 (SH3) domains are mediators of protein-protein interactions. They comprise approximately 60 amino acid residues and are found in many intracellular signaling proteins. Here, we present the crystal structure of the SH3 domain from phosphatidylinositol 3-kinase (PI3K) in complex with the 12-residue proline-rich peptide PD1R (HSKRPLPPLPSL). The crystal structure of the PI3K SH3-PD1R complex at a resolution of 1.7 Å reveals type I ligand orientation of the bound peptide with an extended conformation where the central portion forms a left-handed type II polyproline (PPII) helix. The overall structure of the SH3 domain shows minimal changes on ligand binding. In addition, we also attempted crystallization with another peptide ligand (PD1) where the residue at anchor position P-3 is a tyrosine. The crystals obtained did not contain the PD1 ligand; instead, the ligand binding site is partially occupied by residues Arg18 and Trp55 from the symmetry-related PI3K SH3 molecule. Considering these crystal structures of PI3K SH3 together with published reports, we provide a comparative analysis of protein-ligand interactions that has helped us identify the individual residues which play an important role in defining target specificity.

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.

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