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