Class II Phosphoinositide 3-Kinases Are Downstream Targets of Activated Polypeptide Growth Factor Receptors

ABSTRACT The class II phosphoinositide 3-kinases (PI3K) PI3K-C2α and PI3K-C2β are two recently identified members of the large PI3K family. Both enzymes are characterized by the presence of a C2 domain at the carboxy terminus and, in vitro, preferentially utilize phosphatidylinositol and phosphatidylinositol 4-monophosphate as lipid substrates. Little is understood about how the catalytic activity of either enzyme is regulated in vivo. In this study, we demonstrate that PI3K-C2α and PI3K-C2β represent two downstream targets of the activated epidermal growth factor (EGF) receptor in human carcinoma-derived A431 cells. Stimulation of quiescent cultures with EGF resulted in the rapid recruitment of both enzymes to a phosphotyrosine signaling complex that contained the EGF receptor and Erb-B2. Ligand addition also induced the appearance of a second, more slowly migrating band of PI3K-C2α and PI3K-C2β immunoreactivity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Since both PI3K enzymes can utilize Ca2+ as an essential divalent cation in lipid kinase assays and since the catalytic activity of PI3K-C2α is refractory to the inhibitor wortmannin, these properties were used to confirm the recruitment of each PI3K isozyme to the activated EGF receptor complex. To examine this interaction in greater detail, PI3K-C2β was chosen for further investigation. EGF and platelet-derived growth factor also stimulated the association of PI3K-C2β with their respective receptors in other cells, including epithelial cells and fibroblasts. The use of EGF receptor mutants and phosphopeptides derived from the EGF receptor and Erb-B2 demonstrated that the interaction with recombinant PI3K-C2β occurs through E(p)YL/I phosphotyrosine motifs. The N-terminal region of PI3K-C2β was found to selectively interact with the EGF receptor in vitro, suggesting that it mediates the association of this PI3K with the receptor. However, the mechanism of this interaction remains unclear. We conclude that class II PI3K enzymes may contribute to the generation of 3′ phosphoinositides following the activation of polypeptide growth factor receptors in vivo and thus mediate certain aspects of their biological activity.

[1]  Joseph Schlessinger,et al.  Signal transduction by receptors with tyrosine kinase activity , 1990, Cell.

[2]  Characterization of VPS34, a gene required for vacuolar protein sorting and vacuole segregation in Saccharomyces cerevisiae. , 1990, Molecular and cellular biology.

[3]  T. Pawson,et al.  The tyrosine phosphorylated carboxyterminus of the EGF receptor is a binding site for GAP and PLC‐gamma. , 1990, The EMBO journal.

[4]  M. Ascoli,et al.  Anti-phosphotyrosine immunoprecipitation of phosphatidylinositol 3' kinase activity in different cell types after exposure to epidermal growth factor. , 1990, Biochemical and biophysical research communications.

[5]  A. Ullrich,et al.  Cloning of PI3 kinase-associated p85 utilizing a novel method for expression/cloning of target proteins for receptor tyrosine kinases , 1991, Cell.

[6]  J. Walsh,et al.  Formation of phosphatidylinositol 3-phosphate by isomerization from phosphatidylinositol 4-phosphate. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[7]  W. Gullick,et al.  Prevalence of aberrant expression of the epidermal growth factor receptor in human cancers. , 1991, British medical bulletin.

[8]  L. Stephens,et al.  Pathway of phosphatidylinositol(3,4,5)-trisphosphate synthesis in activated neutrophils , 1991, Nature.

[9]  T. Pawson,et al.  A novel transforming protein (SHC) with an SH2 domain is implicated in mitogenic signal transduction , 1992, Cell.

[10]  A. Ullrich,et al.  Interaction of phosphatidylinositol 3-kinase-associated p85 with epidermal growth factor and platelet-derived growth factor receptors , 1992, Molecular and cellular biology.

[11]  R. Bradshaw,et al.  Activation of phosphatidylinositol 3-kinase by epidermal growth factor, basic fibroblast growth factor, and nerve growth factor in PC12 pheochromocytoma cells. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[12]  S. Volinia,et al.  Phosphatidylinositol 3-kinase: Structure and expression of the 110 kd catalytic subunit , 1992, Cell.

[13]  C. Downes,et al.  Phosphatidylinositol 3-kinase is activated by nerve growth factor and epidermal growth factor in PC12 cells. , 1992, The Journal of biological chemistry.

[14]  P. Hawkins,et al.  Platelet-derived growth factor stimulates synthesis of Ptdlns(3,4,5)P3 by activating a Ptdlns(4,5)P2 3-OH kinase , 1992, Nature.

[15]  T. Pawson,et al.  SH2 domains recognize specific phosphopeptide sequences , 1993, Cell.

[16]  M. Kraus,et al.  Demonstration of ligand-dependent signaling by the erbB-3 tyrosine kinase and its constitutive activation in human breast tumor cells. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[17]  J. Exton,et al.  Activation of the zeta isozyme of protein kinase C by phosphatidylinositol 3,4,5-trisphosphate. , 1993, The Journal of biological chemistry.

[18]  A. Arcaro,et al.  Wortmannin is a potent phosphatidylinositol 3-kinase inhibitor: the role of phosphatidylinositol 3,4,5-trisphosphate in neutrophil responses. , 1993, The Biochemical journal.

[19]  P. Hawkins,et al.  Agonist-stimulated synthesis of phosphatidylinositol(3,4,5)-trisphosphate: a new intracellular signalling system? , 1993, Biochimica et biophysica acta.

[20]  Julian Downward,et al.  Epidermal growth factor regulates p21 ras through the formation of a complex of receptor, Grb2 adapter protein, and Sos nucleotide exchange factor , 1993, Cell.

[21]  K. Takegawa,et al.  Phosphatidylinositol 3-kinase encoded by yeast VPS34 gene essential for protein sorting. , 1993, Science.

[22]  G. Nolan,et al.  Production of high-titer helper-free retroviruses by transient transfection. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[23]  T Pawson,et al.  Interactions between SH2 domains and tyrosine-phosphorylated platelet-derived growth factor beta-receptor sequences: analysis of kinetic parameters by a novel biosensor-based approach , 1993, Molecular and cellular biology.

[24]  C. Heldin,et al.  Identification of two juxtamembrane autophosphorylation sites in the PDGF beta‐receptor; involvement in the interaction with Src family tyrosine kinases. , 1993, The EMBO journal.

[25]  M. Luther,et al.  Involvement of pp60c-src with two major signaling pathways in human breast cancer. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[26]  W. Gullick,et al.  Identification of c‐erbB‐3 binding sites for phosphatidylinositol 3′‐kinase and SHC using an EGF receptor/c‐erbB‐3 chimera. , 1994, The EMBO journal.

[27]  L. Cantley,et al.  Phosphatidylinositol 3‐kinase , 1994, BioEssays : news and reviews in molecular, cellular and developmental biology.

[28]  J. Hancock,et al.  Prenylation of Ras proteins is required for efficient hSOS1-promoted guanine nucleotide exchange. , 1994, The Journal of biological chemistry.

[29]  L. Cantley,et al.  ErbB3 is involved in activation of phosphatidylinositol 3-kinase by epidermal growth factor , 1994, Molecular and cellular biology.

[30]  Andrius Kazlauskas,et al.  PDGF- and insulin-dependent pp70S6k activation mediated by phosphatidylinositol-3-OH kinase , 1994, Nature.

[31]  I Gout,et al.  PI 3‐kinase is a dual specificity enzyme: autoregulation by an intrinsic protein‐serine kinase activity. , 1994, The EMBO journal.

[32]  Andrius Kazlauskas,et al.  The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase , 1995, Cell.

[33]  M. Waterfield,et al.  A family of phosphoinositide 3-kinases in Drosophila identifies a new mediator of signal transduction , 1995, Current Biology.

[34]  D. Baltimore,et al.  Modular binding domains in signal transduction proteins , 1995, Cell.

[35]  M. Zvelebil,et al.  A human phosphatidylinositol 3‐kinase complex related to the yeast Vps34p‐Vps15p protein sorting system. , 1995, The EMBO journal.

[36]  J. Avruch,et al.  Phosphatidylinositol 3-kinase signals activation of p70 S6 kinase in situ through site-specific p70 phosphorylation. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[37]  K. Catt,et al.  Characterization of a soluble adrenal phosphatidylinositol 4-kinase reveals wortmannin sensitivity of type III phosphatidylinositol kinases. , 1996, Biochemistry.

[38]  M. Waterfield,et al.  Binding to the Platelet-derived Growth Factor Receptor Transiently Activates the p85α-p110α Phosphoinositide 3-Kinase Complex in Vivo* , 1996, The Journal of Biological Chemistry.

[39]  L. Williams,et al.  Cpk Is a Novel Class of Drosophila PtdIns 3-Kinase Containing a C2 Domain* , 1996, The Journal of Biological Chemistry.

[40]  Y. Yarden,et al.  ErbB‐2 is a common auxiliary subunit of NDF and EGF receptors: implications for breast cancer. , 1996, The EMBO journal.

[41]  L. Cantley,et al.  p120 Is a Cytosolic Adapter Protein That Associates with Phosphoinositide 3-Kinase in Response to Epidermal Growth Factor in PC12 and Other Cells (*) , 1996, The Journal of Biological Chemistry.

[42]  M. Zvelebil,et al.  Wortmannin inactivates phosphoinositide 3-kinase by covalent modification of Lys-802, a residue involved in the phosphate transfer reaction , 1996, Molecular and cellular biology.

[43]  P. De Camilli,et al.  Phosphoinositides as Regulators in Membrane Traffic , 1996, Science.

[44]  M. Czech,et al.  Mouse p170 Is a Novel Phosphatidylinositol 3-Kinase Containing a C2 Domain* , 1996, The Journal of Biological Chemistry.

[45]  P. Hawkins,et al.  The Gβγ Sensitivity of a PI3K Is Dependent upon a Tightly Associated Adaptor, p101 , 1997, Cell.

[46]  G. Panayotou,et al.  Phosphoinositide 3-kinases: a conserved family of signal transducers. , 1997, Trends in biochemical sciences.

[47]  J. Shipley,et al.  Identification and cDNA cloning of a novel mammalian C2 domain-containing phosphoinositide 3-kinase, HsC2-PI3K. , 1997, Biochemical and biophysical research communications.

[48]  M. Waterfield,et al.  Using structure to define the function of phosphoinositide 3‐kinase family members , 1997, FEBS letters.

[49]  A. Toker,et al.  Signalling through the lipid products of phosphoinositide-3-OH kinase , 1997, Nature.

[50]  P. Cohen,et al.  Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase Bα , 1997, Current Biology.

[51]  M. Zvelebil,et al.  Cloning of a human phosphoinositide 3-kinase with a C2 domain that displays reduced sensitivity to the inhibitor wortmannin. , 1997, The Biochemical journal.

[52]  A. Wong,et al.  Subsets of Epidermal Growth Factor Receptors during Activation and Endocytosis* , 1997, The Journal of Biological Chemistry.

[53]  M. Waterfield,et al.  The CC Chemokine Monocyte Chemotactic Peptide-1 Activates both the Class I p85/p110 Phosphatidylinositol 3-Kinase and the Class II PI3K-C2α* , 1998, The Journal of Biological Chemistry.

[54]  P. Bastiaens,et al.  Binding of a Diphosphotyrosine-containing Peptide That Mimics Activated Platelet-derived Growth Factor Receptor β Induces Oligomerization of Phosphatidylinositol 3-Kinase* , 1998, The Journal of Biological Chemistry.

[55]  Waterfield,et al.  Human PI 3-Kinase C2á - the Role of Calcium and the C2 Domain in Enzyme Activity , 1998 .

[56]  T. Südhof,et al.  C2-domains, Structure and Function of a Universal Ca2+-binding Domain* , 1998, The Journal of Biological Chemistry.

[57]  K. Goto,et al.  A Novel Class II Phosphoinositide 3-Kinase Predominantly Expressed in the Liver and Its Enhanced Expression during Liver Regeneration* , 1998, The Journal of Biological Chemistry.

[58]  A. Lenferink,et al.  Differential endocytic routing of homo‐ and hetero‐dimeric ErbB tyrosine kinases confers signaling superiority to receptor heterodimers , 1998, The EMBO journal.

[59]  N. Copeland,et al.  Cloning and characterization of a novel class II phosphoinositide 3-kinase containing C2 domain. , 1998, Biochemical and biophysical research communications.

[60]  L. Cantley,et al.  Phosphoinositide kinases. , 1998, Annual review of biochemistry.

[61]  L. Pirola,et al.  Bifurcation of lipid and protein kinase signals of PI3Kgamma to the protein kinases PKB and MAPK. , 1998, Science.

[62]  J. Backer,et al.  Regulation of the p85/p110 Phosphatidylinositol 3′-Kinase: Stabilization and Inhibition of the p110α Catalytic Subunit by the p85 Regulatory Subunit , 1998, Molecular and Cellular Biology.

[63]  M. Zvelebil,et al.  Human Phosphoinositide 3-Kinase C2β, the Role of Calcium and the C2 Domain in Enzyme Activity* , 1998, The Journal of Biological Chemistry.

[64]  D. Stern,et al.  Specificity within the EGF family/ErbB receptor family signaling network , 1998, BioEssays : news and reviews in molecular, cellular and developmental biology.

[65]  R. B. Montgomery,et al.  Constitutive Activation of Phosphatidylinositol 3-Kinase by a Naturally Occurring Mutant Epidermal Growth Factor Receptor* , 1998, The Journal of Biological Chemistry.

[66]  S. Volinia,et al.  A Type II Phosphoinositide 3-Kinase Is Stimulated via Activated Integrin in Platelets , 1998, The Journal of Biological Chemistry.