In the present study (14
), Li, Sato and colleagues make the case that the proline-rich tyrosine kinase 2 (Pyk2), a member of the focal adhesion kinase (FAK) family, acts as both a pH sensor and activator of c-Src. Pyk2, a 116-kDa cytoplasmic protein tyrosine kinase, is activated by phosphorylation on tyrosine residues in response to various stimuli, depending on the cell type, including growth factor receptors, chemokine receptors, G protein–coupled receptors, osmotic stress, cell depolarization, and others (19
) (Figure ). For most of these stimuli, activation of Pyk2 requires intracellular calcium release (19
). In contrast to FAK, which is localized to adhesion plaques at the basal side of the cell, Pyk2 is located in the cytosol but can be recruited to plasma membrane, the perinuclear region, or the nucleus in response to different stimuli (20
). Phosphorylation of tyrosine 402 on Pyk2 induces the formation of a complex with the SH2 domain on c-Src (21
), leading to activation of MAPK and JNK signaling pathways (21
Figure 1 Acid-sensing pathway in renal proximal tubular cells. A drop in extracellular fluid pH induces a corresponding decrease in intracellular pH that induces activation of Pyk2, through an unidentified mechanism, by phosphorylation on tyrosine 402. Phosphorylated (more ...)
Li, Sato, et al. (14
) show that Pyk2 is rapidly phosphorylated following exposure of renal epithelial cells to an acidic medium, with peak phosphorylation occurring at 30 seconds after exposure and followed by a persistent low level of increased phosphorylation. Expression of a catalytically inactive dominant-negative Pyk2 prevented the acid-induced activation of NHE3 but had no effect on glucocorticoid-stimulated NHE3 activation. Similarly, suppression of Pyk2 protein by the transfection of cells with a small interfering RNA specific to the opossum mRNA inhibited acid-induced activation of NHE3 without affecting activation by glucocorticoids.
The authors showed that Pyk2 kinase activity and its binding to c-Src are essential for acid-induced c-Src activation. Acid incubation increased the amount of c-Src binding to Pyk2 (14
). Further, in cells expressing the mutant Pyk2Y402F, which was generated to eliminate the c-Src binding site, acid incubation produced no significant activation of NHE3. Finally, expression of a dominant-negative kinase-inactive Pyk2 prevented acid incubation from increasing the tyrosine kinase activity of c-Src.
Then how does the renal epithelial cell sense acid? The authors propose that Pyk2 itself is the sensor (14
). They found that the kinase activity of Pyk2 in vitro is pH dependent, with a 3-fold increase in kinase activity and a nearly 2-fold increase in autophosphorylation activity at pH 7.0 compared with the normal intracellular pH, 7.2. But the devil is in the details. The in vitro experiments were performed at an ATP concentration of 10 μM. The principal effect of pH was to shift the Km
for ATP from 129 μM to 51 μM, concentrations that are both far below the cytosolic ATP concentration. When kinase activity was measured at higher ATP concentrations, the decrease in pH had no effect. Li, Sato, et al. propose the interesting suggestion that the pH dependence of kinase activity may still be physiologically relevant, citing studies by Mandel and colleagues (23
) showing that for the proximal tubule Na,K-ATPase, the apparent Km
for cellular ATP concentration in the intact tubule was much higher than the Km
for ATP concentration of the isolated enzyme. The studies by Mandel et al. are probably not comparable, however, since the Km
for ATP in the Na,K-ATPase is 10-fold higher than that for Pyk2 and because the inhibitors used to alter cellular ATP levels (23
) could have affected Na,K-ATPase activity indirectly.
Second, the authors showed that acid activation of Pyk2 in vitro was inhibited by EGTA, which suggests calcium dependence, although their prior work showed no effect of acid incubation on cell calcium and no effect of the calcium buffer BAPTA (24
), which prevents increases in cell calcium, on acid-induced immediate-early gene expression (16
). Last, experiments in which Pyk2 was overexpressed showed no increase in basal NHE3 activity, as might be expected for a pathway sensitive to small changes in Pyk2 activity. So, although the studies provide compelling evidence that Pyk2 is crucial for the acid-sensing pathway, the elusive acid sensor remains a mystery.