This group has purified p57 and has obtained amino acid sequence from tryptic peptides. Degenerate oligonucleotides from these peptide sequences and from conserved sequences of serine/threonine kinases will be used to obtain PCR- generated DNA products.
In Aim 1, the PCR products will be ligated into pGEM3Zf-vector and the inserts sequenced to verify their difference from known MAP kinases. A unique sequence PCR product will be used to isolate a cDNA for p57 by screening a colon carcinoma cDNA library. Putative cDNA clones will be sequenced on both strands after insertion in both orientations in M13.
In Aim 2, they will determine whether the p57 MBP (myelin basic protein) kinase that is activated by TGFbeta1 in colon carcinoma cells is p57 MAP kinase. Polyclonal anti- peptide antisera to unique sequences (by GenBank analysis) in p57 MAP kinase will be generated and used to selectively remove p57 before analysis of TGFbeta1-modulated MAP kinases.
In Aim 3, they will determine whether Ki-ras, H-ras, PKCbeta, or c-src kinase play roles in p57 activation by transfection experiments. p57 MAP kinase is activated by DAGs in undifferentiated HT29 subclones that have elevated levels of both PKCbeta and c-src kinase. p57 MAP kinase cannot be activated by DAGs in differentiated HT29 subclones which exhibited about 10% of the PKCbeta activity or 20% of the c-src specific activity of undifferentiated subclones. Activities of c-src and PKC will be restored by transfection of expression plasmids for c-src, c-srcY527F, PKCbetaI, or PKCbetaII and p57 activation will be measured in the transfected cells.
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