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.

Phosphatidylinositol 3-Kinase p85 Subunit-Dependent Interaction with BCR/ABL: Molecular Mechanism and Biological Consequences.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2953-2953
Author(s):  
Shuyue Ren ◽  
Elisabeth Bolton ◽  
Tomasz Skorski
Keyword(s):  
Bone Marrow Cells ◽  
Philadelphia Chromosome ◽  
Hematopoietic Cells ◽  
Point Mutations ◽  
Sh3 Domain ◽  
Catalytic Subunit ◽  
Myelogenous Leukemia ◽  
Phosphatidylinositol 3 Kinase ◽  
Sh2 Domains ◽  
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. Mutagenesis approach combined with pull-down assays indicated that both N-terminal and C-terminal SH2 domains (nSH2 and cSH2, respectively) and SH3 domain of p85 play an important role in the interaction with BCR/ABL. SH2 domains exerted their function through binding to the tyrosine-phosphorylated motifs, and SH3 domain recognized proline-rich regions. Disruption of these functions by introduction of point-mutations in nSH2, cSH2 and SH3 domains abrogated their interaction with BCR/ABL. p85 mutant protein (p85-mut) bearing these mutations was not able to interact with BCR/ABL, while its binding to the p110 catalytic subunit of PI-3k was intact. In addition, binding of Shc and Gab2, but not Crk-L, to p85-mut was abrogated. When expressed in BCR/ABL-transformed hematopoietic cells p85-mut diminished activation of Akt kinase, the downstream effector of PI-3k. This effect was associated with the inhibition of BCR/ABL-dependent growth of hematopoeitic cells line and murine bone marrow cells. Interestingly, addition of IL-3 rescued BCR/ABL-transformed cells from the inhibitory effect of p85-mut. SCID mice injected with BCR/ABL-positive hematopoietic cells expressing p85-mut survived longer in comparison to the animals inoculated with BCR/ABL-transformed counterparts transfected with empty plasmid. In conclusion, we have identified the domains of p85 responsible for the interaction with BCR/ABL. Moreover, we demonstrated that expression of p85-mut, which binds to the p110 catalytic subunit of PI-3k but is not able to interact with BCR/ABL, affected the growth ability of BCR/ABL-positive leukemia cells.

Application of FASTTM Fragment-Based Lead Discovery and Structure-Guided Design to Discovery of Small Molecule Inhibitors of BCR-ABL Tyrosine Kinase Active Against the T315I Imatinib-Resistant Mutant.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 698-698 ◽  
Author(s):  
Stephen K. Burley
Keyword(s):  
Tyrosine Kinase ◽  
Fusion Gene ◽  
Clinical Success ◽  
Philadelphia Chromosome ◽  
Myelogenous Leukemia ◽  
Wild Type ◽  
Lead Discovery ◽  
Abl Tyrosine Kinase ◽  
Imatinib Resistant

Abstract The Philadelphia chromosome translocation creates a BCR-ABL fusion gene that encodes a constitutively active BCR-ABL tyrosine kinase, which gives rise to chronic myelogenous leukemia (CML). The clinical success of imatinib (Gleevec) demonstrated that BCR-ABL tyrosine kinase inhibitors can provide effective treatment for CML. However, some CML patients treated with imatinib develop resistance leading to disease progression. The majority of resistance is due to point mutations in BCR-ABL, which give rise to active mutant enzymes that are insensitive to imatinib. In all, ~30 imatinib-resistant BCR-ABL mutants have been identified in clinical isolates. The T315I mutant represents ~20% of clinically observed mutations, making it one of the most common causes of resistance. Second-generation BCR-ABL inhibitors, including AMN-107 and BMS-354825, inhibit many of the clinically relevant mutants but not T315I. Mutant T315I BCR-ABL is, therefore, an important and challenging target for discovery of CML therapeutics. We have applied a proprietary X-ray crystallographic fragment-based lead discovery platform (FASTTM) and structure-guided lead optimization to identify potent inhibitors of wild-type BCR-ABL and the four most common mutants, including T315I. Our lead discovery efforts yielded five chemical series that inhibit both wild-type (WT) and T315I BCR-ABL. Compounds in our most advanced lead series potently inhibit proliferation of K562 cells and Ba/F3 cells with WT BCR-ABL and the four major clinically relevant BCR-ABL mutations (T315I, E255K, M351T, Y253F; see below). Further details describing in vitro and in vivo profiling of these novel BCR-ABL T315I inhibitors will be presented. Ba/F3 cell proliferation for BCR-ABL Inhibitors (EC50, nM) BCR-ABL Form Imatinib AMN-107 BMS-354825 SGX-70430 WT 790 33 12 11 T315I > 10000 > 10000 > 10000 21 Y253F 5700 370 8 334 E255K 8300 350 7 77 M351T 2000 38 28 15 Control Assay Ba/F3 (T315I) + IL3 > 10000 > 10000 > 10000 > 10000

Tyrosine 706 and 807 phosphorylation site mutants in the murine colony-stimulating factor-1 receptor are unaffected in their ability to bind or phosphorylate phosphatidylinositol-3 kinase but show differential defects in their ability to induce early response gene transcription

1991 ◽  
Vol 11 (9) ◽  
pp. 4698-4709
Author(s):  
P van der Geer ◽  
T Hunter
Keyword(s):  
Growth Rate ◽  
Tyrosine Kinase ◽  
Cell Morphology ◽  
Protein Tyrosine Kinase ◽  
Early Response ◽  
Wild Type ◽  
Phosphatidylinositol 3 Kinase ◽  
Protein Tyrosine ◽  
Mutant Receptors ◽  
Phosphatidylinositol 3

The receptor for colony-stimulating factor-1 (CSF-1) is a receptor protein-tyrosine kinase. To study the possible function of CSF-1 receptor autophosphorylation, two autophosphorylation sites, Tyr-706, located in the kinase insert, and Tyr-807, a residue conserved in all protein-tyrosine kinases, were changed independently to either phenylalanine or glycine. Wild-type and mutant receptors were stably expressed in Rat-2 cells. In response to CSF-1, cells expressing Phe- or Gly-706 mutant receptors showed increased growth rate and altered cell morphology. Both the Phe- and Gly-706 mutant receptors associated with and phosphorylated phosphatidylinositol-3 kinase at levels comparable with those of wild-type receptors. However, these mutant receptors differed subtly from each other and from the wild-type receptor in their ability to induce different aspects of the response to CSF-1. The Phe-706 mutant receptor was most strongly affected in its ability to increase growth rate or elevate the levels of c-fos and NGF1A mRNAs, whereas the Gly-706 mutant receptor was most markedly affected in its ability to induce a change in cell morphology or increase the levels of c-jun and NGF1A mRNAs. These findings indicate that Tyr-706 itself, or this region of the receptor, may be important for interaction of the CSF-1 receptor with different signalling pathways. Gly-807 mutant receptors lacked protein-tyrosine kinase activity, failed to respond to CSF-1, and were defective in biosynthetic processing. Phe-807 mutant receptors had 40 to 60% reduced protein-tyrosine kinase activity in vitro. Although cells expressing Phe-807 receptors were able to respond to CSF-1, the changes in growth rate and cell morphology were significantly less than seen with wild-type receptors, and the induction of early response genes was also slightly lower than for the wild-type receptor. In contrast, Phe-807 receptors were equivalent to wild-type receptors when tested for their ability to interact with phosphatidylinositol-3 kinase. These findings indicate that phosphorylation of Tyr-807 may be important for full activation of the receptor.

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.

scholarly journals Tyrosine 706 and 807 phosphorylation site mutants in the murine colony-stimulating factor-1 receptor are unaffected in their ability to bind or phosphorylate phosphatidylinositol-3 kinase but show differential defects in their ability to induce early response gene transcription.

1991 ◽  
Vol 11 (9) ◽  
pp. 4698-4709 ◽  
Author(s):  
P van der Geer ◽  
T Hunter
Keyword(s):  
Growth Rate ◽  
Tyrosine Kinase ◽  
Cell Morphology ◽  
Protein Tyrosine Kinase ◽  
Early Response ◽  
Wild Type ◽  
Phosphatidylinositol 3 Kinase ◽  
Protein Tyrosine ◽  
Mutant Receptors ◽  
Phosphatidylinositol 3

The receptor for colony-stimulating factor-1 (CSF-1) is a receptor protein-tyrosine kinase. To study the possible function of CSF-1 receptor autophosphorylation, two autophosphorylation sites, Tyr-706, located in the kinase insert, and Tyr-807, a residue conserved in all protein-tyrosine kinases, were changed independently to either phenylalanine or glycine. Wild-type and mutant receptors were stably expressed in Rat-2 cells. In response to CSF-1, cells expressing Phe- or Gly-706 mutant receptors showed increased growth rate and altered cell morphology. Both the Phe- and Gly-706 mutant receptors associated with and phosphorylated phosphatidylinositol-3 kinase at levels comparable with those of wild-type receptors. However, these mutant receptors differed subtly from each other and from the wild-type receptor in their ability to induce different aspects of the response to CSF-1. The Phe-706 mutant receptor was most strongly affected in its ability to increase growth rate or elevate the levels of c-fos and NGF1A mRNAs, whereas the Gly-706 mutant receptor was most markedly affected in its ability to induce a change in cell morphology or increase the levels of c-jun and NGF1A mRNAs. These findings indicate that Tyr-706 itself, or this region of the receptor, may be important for interaction of the CSF-1 receptor with different signalling pathways. Gly-807 mutant receptors lacked protein-tyrosine kinase activity, failed to respond to CSF-1, and were defective in biosynthetic processing. Phe-807 mutant receptors had 40 to 60% reduced protein-tyrosine kinase activity in vitro. Although cells expressing Phe-807 receptors were able to respond to CSF-1, the changes in growth rate and cell morphology were significantly less than seen with wild-type receptors, and the induction of early response genes was also slightly lower than for the wild-type receptor. In contrast, Phe-807 receptors were equivalent to wild-type receptors when tested for their ability to interact with phosphatidylinositol-3 kinase. These findings indicate that phosphorylation of Tyr-807 may be important for full activation of the receptor.

scholarly journals Dominant missense mutations in a novel yeast protein related to mammalian phosphatidylinositol 3-kinase and VPS34 abrogate rapamycin cytotoxicity.

1993 ◽  
Vol 13 (10) ◽  
pp. 6012-6023 ◽  
Author(s):  
R Cafferkey ◽  
P R Young ◽  
M M McLaughlin ◽  
D J Bergsma ◽  
Y Koltin ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Dna Sequence ◽  
Leucine Zipper ◽  
Sequence Similarity ◽  
Missense Mutations ◽  
Wild Type ◽  
Phosphatidylinositol 3 Kinase ◽  
Haploid Strain ◽  
Diploid Cells ◽  
Phosphatidylinositol 3

Rapamycin is a macrolide antifungal agent that exhibits potent immunosuppressive properties. In Saccharomyces cerevisiae, rapamycin sensitivity is mediated by a specific cytoplasmic receptor which is a homolog of human FKBP12 (hFKBP12). Deletion of the gene for yeast FKBP12 (RBP1) results in recessive drug resistance, and expression of hFKBP12 restores rapamycin sensitivity. These data support the idea that FKBP12 and rapamycin form a toxic complex that corrupts the function of other cellular proteins. To identify such proteins, we isolated dominant rapamycin-resistant mutants both in wild-type haploid and diploid cells and in haploid rbp1::URA3 cells engineered to express hFKBP12. Genetic analysis indicated that the dominant mutations are nonallelic to mutations in RBP1 and define two genes, designated DRR1 and DRR2 (for dominant rapamycin resistance). Mutant copies of DRR1 and DRR2 were cloned from genomic YCp50 libraries by their ability to confer drug resistance in wild-type cells. DNA sequence analysis of a mutant drr1 allele revealed a long open reading frame predicting a novel 2470-amino-acid protein with several motifs suggesting an involvement in intracellular signal transduction, including a leucine zipper near the N terminus, two putative DNA-binding sequences, and a domain that exhibits significant sequence similarity to the 110-kDa catalytic subunit of both yeast (VPS34) and bovine phosphatidylinositol 3-kinases. Genomic disruption of DRR1 in a mutant haploid strain restored drug sensitivity and demonstrated that the gene encodes a nonessential function. DNA sequence comparison of seven independent drr1dom alleles identified single base pair substitutions in the same codon within the phosphatidylinositol 3-kinase domain, resulting in a change of Ser-1972 to Arg or Asn. We conclude either that DRR1 (alone or in combination with DRR2) acts as a target of FKBP12-rapamycin complexes or that a missense mutation in DRR1 allows it to compensate for the function of the normal drug target.

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