P57 MAP KINASE

描述：（申请人的摘要）：该小组已经纯化了p57， 从胰蛋白酶肽中获得了氨基酸序列。 简并 寡核苷酸从这些肽序列和保守的 丝氨酸/苏氨酸激酶的序列将用于获得PCR。 产生DNA产物。 在目标1中，将PCR产物连接到 pGEM 3 Zf-载体和插入物测序以验证它们与 已知的MAP激酶。 一个独特的序列PCR产物将用于 通过筛选结肠癌cDNA文库分离p57 cDNA。 插入后，将对推定的cDNA克隆的两条链进行测序 在M13的两个方向上。 在目标2中，他们将确定 p57 MBP（髓鞘碱性蛋白）激酶，由TGF β 1激活， 结肠癌细胞p57 MAP激酶。 多克隆抗肽 针对p57 MAP激酶中独特序列的抗血清（通过GenBank分析） 将产生并用于在分析p57之前选择性地去除p57。 TGF β 1调节的MAP激酶。 在目标3中，他们将确定 Ki-ras、H-ras、PKC β或c-src激酶在p57激活中发挥作用， 转染实验。 p57 MAP激酶被DAGs激活， 未分化的HT 29亚克隆具有升高的水平， PKC β和c-src激酶。 p57 MAP激酶不能被DAG激活 在分化的HT 29亚克隆中，显示出约10%的 PKC β活性或20%的c-src比活性 未分化的亚克隆 c-src和PKC的活动将在 通过转染c-src，c-srcY 527 F， PKC β I或PKC β II和p57激活将在 转染细胞

DESCRIPTION: (Applicant's Abstract): 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.