ANALYSIS OF NOVEL PLECKSTRIN HOMOLOGY DOMAIN

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Phosphatidylinositol 3-kinase (PI3K) is an important enzyme in signal transduction pathways. PI3K generates the second messengers PI(3,4)P2 and PI(3,4,5)P3, which mediate responses by binding to pleckstrin homology (PH) domains in downstream signaling molecules. This application proposes to identify the roles of two new 3-phosphoinositides (3-PI) binding proteins that the investigators identified in PI3K signaling. Spiel is a PH domain containing protein that has an N-terminal SH2 and a C-terminal PH domain with a tyrosine (tyr) phosphorylation site located between these domains. Spiel likely functions as an adaptor molecule to couple proteins bound to its SH2 domain and/or phospho-tyrosine to 3-PIs in stimulated cells via its PH domain. The investigators plan: (1) To determine the mechanism whereby expression of Spiel (Y139F) in which the tyr phosphorylation site (Y139) is mutated to alanine enhances activation of AKT in response to pervanadate stimulation, and the physiological stimuli that lead to Spiel tyrosine phosphorylation. The investigators will test whether Spiel interacts via pY139 with proteins that negatively regulate signaling in B cells, determine cellular localization of Spiel in stimulated cells and whether Spiel localizes SHIP/SHP/Cbl to plasma membrane (PM), whether Spiel (Y139F) acts pre- or post- generation of Ptdins (3,4,5) P3 to enhance AKT activation and whether overexpression of Spiel (Y139F) enhances activation of signaling molecules upstream of PI3K and AKT activation. In addition, the investigators propose to identify the upstream signaling system that leads to Spiel tyr phosphorylation. (2) The investigators propose to identify: A) tyr phosphorylated proteins that bind the SH2 domain of Spiel and (B) SH2 domain containing proteins that bind pY139 on Spiel. Once proteins are identified, the investigators will test (C) their in vivo relevance to Spiel signaling and (D) their mode of regulation. (E) The investigators hypothesize that PIP7, a second new PH domain containing protein that the investigators identified that binds 3-PIs with high affinity, functions as an adaptor molecule in cells, and the investigators will identify proteins that bind PIP7 and determine their relevance to PIP7 and PI3 kinase signaling as for Spiel. (3) To determine the role for Spiel in development and identify signaling pathways in which Spiel plays an essential function, the investigators will create mice nullizygous for Spiel. The investigators will analyze Spiel -/- mice by determining whether their B and T cell development is normal; whether they have normal serum Ig and humoral responses; and whether the T and B cells isolated from these animals function normally following stimulation. Spiel -/- cell and cell lines will be used to definitely confirm or refute essential roles for Spiel in mediating the functions identified in previous aims.