Abstract
PYK2/CAKbeta is a recently described cytoplasmic tyrosine kinase related to p125 focal adhesion kinase (p125(FAK)) that can be activated by a number of stimuli including growth factors, lipids, and some G protein-coupled receptors. Studies suggest PYK2/CAKbeta may be important for coupling various G protein-coupled receptors to the mitogen-activated protein kinase (MAPK) cascade. The hormone neurotransmitter cholecystokinin (CCK) is known to activate both phospholipase C-dependent cascades and MAPK signaling pathways; however, the relationship between these remain unclear. In rat pancreatic acini, CCK-8 (10 nM) rapidly stimulated tyrosine phosphorylation and activation of PYK2/CAKbeta by both activation of high affinity and low affinity CCK(A) receptor states. Blockage of CCK-stimulated increases in protein kinase C activity or CCK-stimulated increases in [Ca(2+)](i), inhibited by 40-50% PYK2/CAKbeta but not p125(FAK) tyrosine phosphorylation. Simultaneous blockage of both phospholipase C cascades inhibited PYK2/CAKbeta tyrosine phosphorylation completely and p125(FAK) tyrosine phosphorylation by 50%. CCK-8 stimulated a rapid increase in PYK2/CAKbeta kinase activity assessed by both an in vitro kinase assay and autophosphorylation. Total PYK2/CAKbeta under basal conditions was largely localized (77 +/- 7%) in the membrane fraction, whereas total p125(FAK) was largely localized (86 +/- 3%) in the cytosolic fraction. With CCK stimulation, both p125(FAK) and PYK2/CAKbeta translocated to the plasma membrane. Moreover CCK stimulation causes a rapid formation of both PYK2/CAKbeta-Grb2 and PYK2/CAKbeta-Crk complexes. These results demonstrate that PYK2/CAKbeta and p125(FAK) are regulated differently by CCK(A) receptor stimulation and that PYK2/CAKbeta is probably an important mediator of downstream signals by CCK-8, especially in its ability to activate the MAPK signaling pathway, which possibly mediates CCK growth effects in normal and neoplastic tissues.
Highlights
proline-rich tyrosine kinase 2 (PYK2)/CAK is a recently described cytoplasmic tyrosine kinase related to p125 focal adhesion kinase (p125FAK) that can be activated by a number of stimuli including growth factors, lipids, and some G proteincoupled receptors
When pancreatic acini were incubated with 10 nM CCK-8 for 2.5 min (a concentration and time that causes a maximal stimulation of tyrosine phosphorylation of p125FAK and paxillin in previous studies [17, 18]), immunoprecipitated with anti-phosphotyrosine monoclonal antibody (mAb) (PY20), and analyzed by Western blotting with the same antibody (Fig. 1, left upper panel), an increase in the tyrosine phosphorylation of at least two major components of molecular mass 115–140 kDa and 65– 80 kDa was seen
Whether an anti-phosphotyrosine mAb is used for immunoprecipitation followed by protein identification using an anti-PYK2/CAK mAb or the same mAbs are used in reverse order, CCK-8 caused a marked increased in the tyrosine phosphorylation of a single PYK2/CAK immunoreactive band
Summary
PYK2/CAK is a recently described cytoplasmic tyrosine kinase related to p125 focal adhesion kinase (p125FAK) that can be activated by a number of stimuli including growth factors, lipids, and some G proteincoupled receptors. Recent studies (30 –34) suggest that with activation of some GPCRs [30, 32,33,34,35], which cause increases in cellular calcium as well as some integrins [31, 36, 37], growth factors [37,38,39], or phospholipids [32, 40], one protein, the cytoplasmic tyrosine kinase proline-rich kinase 2 (PYK2) [30] ( called cell adhesion kinase  (CAK) [41], related adhesion focal tyrosine kinase (RAFTK) [42], focal adhesion kinase 2 (FAK2), and calcium-dependent tyrosine kinase (CADTK) [40]) may be a important tyrosine-phosphorylated substrate responsible for coupling these intracellular cascades. With different GPCRs, the coupling to PYK2/CAK and the mechanisms of the possible coupling of these different intracellular cascades vary widely At present, it is unknown whether CCKA receptor activation stimulates tyrosine phosphorylation of PYK2/CAK or whether its ability to activate one or both limbs of the PLC cascade is important for this tyrosine phosphorylation if it occurs. The ability of CCKA receptor activation to stimulate PYK2/CAK tyrosine phosphorylation and its relationship with these other intracellular signaling cascades was explored in pancreatic acini, which are one of the main physiological sites of action of CCK
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