Abstract
The focal adhesion kinases, p125FAK and proline-rich kinase 2 (PYK2), are involved in numerous processes as adhesion, cytoskeletal changes, and growth. These kinases have 45% homology and share three tyrosine phosphorylation (TyrP) sites. Little information exists on the ability of stimulants to cause TyrP of each kinase site and the cellular mechanism involved. We explored the ability of the neurotransmitter/hormone, CCK, to stimulate TyrP at each site. In rat pancreatic acini, CCK stimulated TyrP at each site in both kinases. TyrP was rapid except for pY397FAK. The magnitude of TyrP differed with the different FAK and PYK2 sites. The CCK dose-response curve for TyrP for sites in each kinase was similar. CCK-JMV, an agonist of the high affinity receptor state and antagonist of the low affinity receptor state, was less efficacious than CCK at each FAK/PYK2 site and inhibited CCK maximal stimulation. Thapsigargin decreased CCK-stimulated TyrP of pY402PYK2 and pY925FAK but not the other sites. GF109203X reduced TyrP of only the PYK2 sites, pY402 and pY580. GF109203X with thapsigargin decreased TyrP of pY402PYK2 and the three FAK sites more than either inhibitor alone. Basal TyrP of pY397FAK was greater than other sites. These results demonstrate that CCK stimulates tyrosine phosphorylation of each of the three homologous phosphorylation sites in FAK and PYK2. However, CCK-stimulated TyrP at these sites differs in kinetics, magnitude, and participation of the high/low affinity receptor states and by protein kinase C and [Ca2+]i. These results show that phosphorylation of these different sites is differentially regulated and involves different intracellular mechanisms in the same cell.
Highlights
The closely related nonreceptor focal adhesion tyrosine kinases (FAK)1 p125FAK and PYK2 transduce key extracellular signals that are involved in mediating growth, adhesion, cy
Maximal phosphorylation for all three PYK2 phosphorylation sites, i.e. pY402, pY580, and pY881 was reached after 0.5–1 min as it was for tyrosine phosphorylation of the FAK phosphorylation sites pY577 and pY925 (Fig. 1 and Table I)
Recent studies show that CCKA receptor activation, similar to a number of growth factors, oncogenes, integrins, and stimulation of some other G protein-coupled receptors, causes activation and tyrosine phosphorylation of the two related kinases p125FAK and PYK2 [2, 9, 11, 19, 34, 38, 39]
Summary
The closely related nonreceptor focal adhesion tyrosine kinases (FAK) p125FAK and PYK2 transduce key extracellular signals that are involved in mediating growth, adhesion, cy-. Little information exists on the comparative ability (kinetics and magnitude) of a given stimulant to increase tyrosine phosphorylation of the three homologous sites in these two kinases or the participation of other cellular cascades such as the two limbs of the phospholipase C cascade in this stimulation at a given site. It has been shown in previous studies that in rat pancreatic acini the activation of the CCKA receptor leads to a rapid tyrosine phosphorylation of PYK2 and FAK [9, 19]. CCK-stimulated FAK/PYK2 Phosphospecific Phosphorylation one of its main physiological stimulants [25, 26], this cell system is an ideal model to investigate the comparative ability of a given stimulant to cause site-specific differences in tyrosine phosphorylation of FAK and PYK2 in the same cell and study the intracellular mechanisms involved in the stimulation of tyrosine phosphorylation of these phosphospecific sites of these two kinases
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