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.
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