P57 MAP KINASE

描述：(申请人摘要)：此组已提纯p57和 已经从胰蛋白酶多肽中获得了氨基酸序列。堕落 来自这些肽序列和来自保守序列的寡核苷酸 丝氨酸/苏氨酸激酶序列将被用来获得 产生了DNA产物。在目标1中，聚合酶链式反应产物将连接到 对pGEM3Zf载体和插入片段进行测序，以验证它们与 已知的地图激活酶。一种独特的序列聚合酶链式反应产物将用于 通过筛选结肠癌c DNA文库分离p57的c DNA。 插入后，假定的cdna克隆将在两条链上进行测序。 在M13的两个方向上。在目标2中，他们将决定是否 P57 MBP(髓鞘碱性蛋白)激酶被转化生长因子β1激活 结肠癌细胞是p57的MAP激酶。多克隆抗肽 抗p57 MAP激酶独特序列的抗血清(GenBank分析) 将生成并用于在分析之前选择性地删除p57 TGFbeta1调控的MAP激酶。在目标3中，他们将决定是否 Ki-ras、H-ras、PKCbeta或c-src激酶通过以下途径在p57激活中发挥作用 转基因实验。P57 MAP激酶被DAG激活 未分化的HT29亚克隆，两者的水平都升高 PKCβ和c-src激酶。P57蛋白激酶不能被DAG激活 在分化的HT29亚克隆中，表现出约10%的 PKCβ活性或c-src比活性的20% 未分化的亚克隆。C-src和PKC的活动将是 通过c-src、c-srcY527F、 PKCbetaI或PKCbetaII和p57的激活将在 转基因细胞。

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.