The human c-fes proto-oncogene encodes a 93 kDa cytoplasmic tyrosine kinase (Fes) involved in hematopoiesis, angiogenesis, and embryonic development. Transfection of an immature myeloid leukemia cell line (K-562) with Fes results in growth suppression and terminal differentiation, identifying Fes as a key regulator of myeloid cell growth and a rational target for the differentiation therapy of myeloid leukemia. Structurally, Fes consists of a unique N-terminal region, a Src homology 2 (SH2) domain, and a C-terminal kinase domain. Four hypotheses regarding the role of each domain in the regulation of kinase activity, interaction with downstream effectors, and biological function will be tested: 1) The Fes N-terminal region contains a novel protein-protein interaction domain essential for the recognition of BCR and other substrates. The specific region of the Fes N-terminal domain responsible for binding to BCR, a regulator of Rho-family small GTPases and Fes substrate, will be mapped in vitro. The biological significance of the BCR-binding domain will be assessed by testing deletion, insertion, and substitution mutants of this region in the K-562 cell differentiation model. 2) The Fes SH2 domain is essential for protein-protein interaction during differentiation signaling. Chimeric Fes proteins containing the SH2 domains of other signaling molecules will be tested in the K-562 differentiation model. Demonstration that the chimeras show diminished biological activity while maintaining kinase activity and correct subcellular localization will strongly support a role for the Fes SH2 domain in specific protein-protein interactions. 3) The SH2-containing proteins Vav, STAT-3, and PI3K are differentiation-related Fes substrates. Preliminary data implicate these proteins as downstream Fes effectors.
This Aim will test whether Fes interacts with these proteins during myeloid differentiation, and determine whether Fes kinase domain Tyr autophosphorylation sites bind to the SH2 domains of these molecules in vitro and in vivo. 4) Activation of Fes requires oligomerization and transphosphorylation. Preliminary data show that active Fes is an oligomer and autophosphorylates via an intermolecular mechanism. Mutagenesis experiments will test whether an N-terminal coiled-coil domain identified by computer analysis is required for oligomerization and biological activity. In complementary experiments, autophosphorylation-defective mutants of Fes will be tested for dominant-negative activity. Kinase-inactive Fes mutants are predicted to suppress biological responses dependent upon endogenous Fes activation by non-productive oligomerization events.
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