Multiple Proline-rich Regions of GAP-associated Phosphoprotein p62 Bind with Different Affinities to the Src Homology 3 Domains of Fyn and Src

Several proteins of Jurkat cells were identified on SDS-PAGE gels by Coomassie Blue staining that bound specifically to affinity matrices made of five different Src homology 3 (SH3) domains fused to glutathione S-transferase (GST). Purification of the major specific band of approximately 70kDa with affinity beads of the SH3 domain of Fyn tyrosine kinase resulted in an identification of a GAP-associated p62-related protein as a ligand to the Fyn and Src SH3 domains. Indeed, from a lysate of a Rous sarcoma virus-transformed rat fibroblast line, Src co-precipitated with the 70kDa and also bound to a puta-tive SH3 binding sequence of p62. Bacterially expressed GST fusion proteins containing sequences encompassing each of the proline-rich putative SH3 binding sites of p62 bound to a subset of SH3 domains with different affinities. Phos-pholipase Cgannma 2-SH3 also revealed strong binding to the bacterially expressed p62 fusion proteins in vitro but did not show primary binding to the cellular 70kDa. The multiple SH3 binding sequences with different affinities to various SH3 mole-cules together with their phosphorylation on tyrosine residue(s) suggest a role of p62 as a foothold on which signal transduction proteins, including Src-family kinases, link together

Partial Catalytic Domains of New Protein-tyrosine Kinases Cloned from cDNA Amplified by Polymerase Chain Reaction

A feature common to all members of the protein-tyrosine kinase (PTK) family is a highly conserved catalytic domain which is characteristic of this group. Degenerate oligonucleotide primers corresponding to two of the most highly conserved regions of the PTK catalytic domain were designed to amplify cDNA sequences of restricted subfamilies of PTKs from rat brain mRNA in the polymerase chain reaction (PCR). A third degenerate oligonucleotide primer corresponding to a highly conserved, PTK subfamily-specific sequence located between the two sequences mentioned above was also used to amplify cDNA sequences of PTKs of novel subfamilies from rat brain mRNA. pBluescript PCR libraries were constructed from the PCR-amplified cDNA. The PCR libraries were then screened by DNA sequencing for PTK-related sequences. Several sequences were identified that, on the basis of sequence comparison with known PTKs in GenBank, may encode new PTKs

Cloning of a cDNA for the Human Cell Adhesion Kinase β

Cell adhesion kinase beta (CAKbeta) is the second protein-tyrosine kinase (PTK) of the focal adhesion kinase (FAK) subfamily with large N- and C-domains in addition to the central kinase domain but without Src homology 2 and 3 (SH-2 and SH-3) domains. In this paper, cloning and sequencing of a cDNA encoding human CAKβ are described. A full-length clone (clone B) contained 4,157- base pairs of human CAKβ cDNA including 243-base pairs of the 5\u27-untranslated sequence and 881-base pairs of the 3\u27-untranslated sequence with a polyadenyla-tion signal (ATTAAA). The clone B of human CAKβ cDNA has an open read-ing frame encoding 1009 amino acid residues ; the human CAKβ has the same number of amino acid residues in the N-, C-, and kinase-domains as rat CAKβ . The amino acid sequence of human CAKβ is 95.4% identical with that of rat CAKβ . The species difference is most prominent in the C-domain. All three previously-recognized, subfamily-specific residues in the kinase domains of FAK and the rat CAKβ are also found in the human CAKβ . The residues V??? and A???, which have been considered to be characteristic to CAKβ , are found to be conserved also in the human CAK? . It has been postulated that CAKβ is important as a docking protein. The autophosphorylation site and also the ligand site to the SH-2 domains of the Src-family PTKs, Y???AEI, are found to be conserved in the human CAKβ . The ligand sequence for the Grb2 SH-2 domain, Y???HNV of the rat CAKβ , is found functionally conserved in the human CAKβ , Y???LNV. The third ligand sequence, E???PPPKPSR, participating in the binding to the SH-3 domains of pp130cas and Efs, is also found conserved in the human CAKβ . The extreme N- terminal 88 amino acid residues of the rat CAKβ were previously found entirely different from FAK and found unique to CAK? . Ninety four percent of those 88 residues in the human CAKβ are found identical with the rat CAKβ . This high sequence homology strongly suggeststhat this region is involved in the specific function of CAKβ different from FAK

CAKβ/Pyk2 Kinase Is a Signaling Link for Induction of Long-Term Potentiation in CA1 Hippocampus

AbstractLong-term potentiation (LTP) is an activity-dependent enhancement of synaptic efficacy, considered a model of learning and memory. The biochemical cascade producing LTP requires activation of Src, which upregulates the function of NMDA receptors (NMDARs), but how Src becomes activated is unknown. Here, we show that the focal adhesion kinase CAKβ/Pyk2 upregulated NMDAR function by activating Src in CA1 hippocampal neurons. Induction of LTP was prevented by blocking CAKβ/Pyk2, and administering CAKβ/Pyk2 intracellularly mimicked and occluded LTP. Tyrosine phosphorylation of CAKβ/Pyk2 and its association with Src was increased by stimulation that produced LTP. Finally, CAKβ/Pyk2-stimulated enhancement of synaptic AMPA responses was prevented by blocking NMDARS, chelating intracellular Ca2+, or blocking Src. Thus, activating CAKβ/Pyk2 is required for inducing LTP and may depend upon downstream activation of Src to upregulate NMDA receptors