CELLULAR BASIS OF ACTION OF GASTROINTESTINAL PEPTIDES

Bombesin和cck相关肽广泛存在于胃肠道和中枢神经系统，然而，它们的细胞作用基础方面仍不清楚，特别是酪氨酸磷酸化在其信号级联中的作用。在这一年中，我们主要关注CCKA-R激活引起的酪氨酸磷酸化。在先前的研究中，CCK-A-R的激活已被证明与磷脂酶C的激活耦合，通过动员细胞[Ca-2+]，以及PKC， PLD和PLA-2的激活。我们和其他人最近的研究表明，酪氨酸磷酸化的增加也可能是一个重要的细胞级联反应。我们已经证明CCK-A-R引起p125黏附激酶、paxillin和p130-cas的酪氨酸磷酸化。我们发现CCKA-R激活导致新的细胞质酪氨酸激酶PYK2/CAKB的酪氨酸磷酸化。该激酶被许多生长因子和生物活性脂质激活，是MAP激酶级联联的重要细胞机制。CCKA-R激活导致PYK-2/CAKB酪氨酸快速磷酸化，这依赖于[Ca2+]i和PKC激活的变化，并导致PYK-2激酶活性增加。CCKA-R受体的高亲和力和低亲和力状态均刺激PYK-2酪氨酸磷酸化。CCKA-R PYK2酪氨酸磷酸化需要肌动蛋白细胞骨架的完整性，而不需要微管网络。CCKA-R激活导致PYK-2易位至质膜，形成PYK-2- crk复合物和PYK-2Grb复合物。这些结果表明，PYK-2的激活可能是CCK刺激MAPK信号通路能力的重要介质，MAPK信号通路被认为介导了CCK引起的许多生长效应。

Bombesin- and CCK-related peptides are found widely in the gastrointestinal (GI) tract and central nervous system, however, aspects of their cellular basis of action remain unclear, particularly the role of tyrosine phosphorylation in their signaling cascade. During this year we have primarily focused on tyrosine phosphorylation caused by activation of CCKA-R. In previous studies CCK-A-R activation has been shown to be coupled to activation of phospholipase C with mobilization of cellular [Ca-2+], and activation of PKC, PLD, and PLA-2. Recent studies by us and others show increased tyrosine phosphorylation may also be an important cellular cascade. We have demonstrated CCK-A-R causes tyrosine phosphorylation of p125 focal adhesion kinase, paxillin, and p130-cas. We have found that CCKA-R activation causes tyrosine phosphorylation of the novel cytoplasmic tyrosine kinase, PYK2/CAKB. This kinase is activated by a number of growth factors and bioactive lipids and is an important cellular mechanism for coupling to the MAP kinase cascade. CCKA-R activation caused rapid PYK-2/CAKB tyrosine phosphorylation which was dependent on changes in [Ca2+]i and PKC activation and resulted in an increase in PYK-2 kinase activity. Both high and low affinity states of the CCKA-R receptor stimulated PYK-2 tyrosine phosphorylation. CCKA-R PYK2 tyrosine phosphorylation required the integrity of the actin cytoskeleton but not the microtubule network. CCKA-R activation caused PYK-2 translocation to the plasma membrane and formation of PYK-2-CRK complexes and PYK-2Grb complexes. These results demonstrate that activation of PYK-2 is likely an important mediator of the ability of CCK to stimulate the MAPK signaling pathway which is thought to mediate many of the growth effects caused by CCK.