A noncatalytic domain conserved among cytoplasmic protein-tyrosine kinases modifies the kinase function and transforming activity of Fujinami sarcoma virus P130gag-fps.

Abstract

Proteins encoded by oncogenes such as v-fpslfes, v-src, v-yes, v-abl, and v-fgr are cytoplasmic protein tyrosine kinases which, unlike transmembrane receptors, are localized to the inside of the cell. These proteins possess two contiguous regions of sequence identity: a C-terminal catalytic domain of 260 residues with homology to other tyrosine-specific and serine-threonine-specific protein kinases, and a unique domain of approximately 100 residues which is located N terminal to the kinase region and is absent from kinases that span the plasma membrane. In-frame linker insertion mutations in Fujinami avian sarcoma virus which introduced dipeptide insertions into the most stringently conserved segment of this N-terminal domain in P130"w-lps impaired the ability of Fujinami avian sarcoma virus to transform rat-2 cells. The P130gag-s proteins encoded by these transformation-defective mutants were deficient in protein-tyrosine kinase activity in rat cells. However v-fps polypeptides derived from the mutant Fujinami avian sarcoma virus genonles and expressed in Escherichia coli as trpE-v-fps fusion proteins displayed essentially wild-type enzymatic activity, even though they contained the mutated sites. Deletion of the N-terminal domain from wild-type and mutant v-frs bacterial proteins had little effect on autophosphorylating activity. The conserved N-terminal domain of P130EagPS is therefore not required for catalytic activity, but can profoundly influence the adjacent kinase region. The presence of this noncatalytic domain in all known cytoplasmic tyrosine kinases of higher and lower eucaryotes argues for an important biological function. The relative inactivity of the mutant proteins in rat-2 cells compared with bacteria suggests that the noncatalytic domain may direct specific interactions of the enzymatic region with cellular components that regulate or mediate tyrosine kinase function.

Signal transduction in mammary tumorigenesis: a transgenic perspective

“…The primary function of RTK tyrosine phosphorylation is to generate binding sites for cytoplasmic or plasma membrane associated proteins involved in transducing proliferative or di erentiating signals to the nucleus. These`signaling proteins' each contain modular Src homology 2 (SH2) (Sadowski et al, 1986) or protein tyrosine binding/ interacting (PTB/PID) (van der Geer et al, 1995) domains and directly interact with phosphotyrosyl proteins in a sequence speci®c manner (reviewed in Pawson and Scott, 1997;Sudol 1998). Such signaling proteins can be loosely grouped into two classes: enzymes and non-enzymatic`adapter' proteins.…”

Deletions in the SH2 domain of p60v-src prevent association with the detergent-insoluble cellular matrix.

“…Two regions of sequence conservation have been identified in the amino termini of cytoplasmic tyrosine kinases, including p6Osrc (32,36). These regions are SH2 (src homology 2, residues 137 through 241 in src; see Fig. lA) and SH3 (src homology 3, residues 84 through 114).…”

“…SH2 sequences are also present in phospholipase C--y (39), GAP (ras GTPase-activating protein) (43), and the crk oncogene product (32). Mutations of the SH2 regions of v-src and v-fps produce transformation-defective or temperature-sensitive tyrosine kinase proteins (2,5,25,26,36,44). Point mutations in the SH2 region of the proto-oncogene p60c-src have been shown to elevate kinase activity and transforming ability (17,33).…”

Structure and regulation of Src family kinases

“…The discovery of the SH2 domain as a functionally important, but noncatalytic segment of B100 residues conserved in v-Fps, Abl, and Src kinases (Sadowski et al, 1986) gave rise to the concept of 'modular signaling domain' (Pawson, 1995). Studies of the function and specificity of the SH2 and SH3 domains of Src and of their effect on the activity of the adjacent kinase domain have provided a powerful paradigm for the functional dissection of a host of modular signaling domains (see Pawson (2004) for an excellent review and historical perspective).…”