Abstract
It is generally thought that activation of phospholipase Cbeta (PLCbeta) by Galphaq accounts for most of the effects of Gq-coupled receptors. Here we describe a novel effect of Galphaq that is independent of the PLCbeta pathway. Expression of the constitutively active Galphaq mutant Galphaq(Q209L) promoted an increase in glycogen synthase kinase-3beta (GSK-3beta) activity that was associated with increased phosphorylation of Tyr216 on GSK-3beta. Galphaq(Q209L)-AA, a mutant that cannot activate PLCbeta, also induced GSK-3beta activation and phosphorylation of Tyr216. We speculate that the protein-tyrosine kinase Csk (C-terminal Src kinase), which is also activated by Galphaq(Q209L) and Galphaq(Q209L)-AA, acts upstream of GSK-3beta. Expression of Csk accentuated the activation of GSK-3beta by Galphaq(Q209L), whereas catalytically inactive Csk blocked GSK-3beta activation by Galphaq(Q209L). Recombinant Csk phosphorylated and activated GSK-3beta in vitro, and GSK-3beta coprecipitated with Csk from cell lysates. These results suggest that activation of Csk and GSK-3beta by Galphaq may contribute to the physiological and pathological effects of Gq-coupled receptors.
Highlights
It is generally thought that activation of phospholipase C (PLC) by G␣q accounts for most of the effects of Gq-coupled receptors
G␣q(Q209L) Effects on Akt and Glycogen synthase kinase-3 (GSK-3) Are PLC-independent—In an earlier study we suggested that inhibition of phosphatidylinositol 3-kinase (PI3K)/ Akt by activated G␣q is independent of the canonical PLC pathway, based on its resistance to U73122 [20]
We found that activated G␣q inhibits PI3K/Akt signaling via a mechanism that is independent of PLC activation and that might involve an inhibitory interaction between G␣q and p110␣ PI3K (Fig. 1A and Ref. 20)
Summary
PLC, phospholipase C; GSK-3, glycogen synthase kinase-3; Csk, C-terminal Src kinase; Csk-KD, kinasedead Csk; HA, hemagglutinin; HEK, human embryonic kidney; LPA, lysophosphatidic acid; PI3K, phosphatidylinositol 3-kinase; PYK2, proline-rich protein kinase 2; Btk, Bruton’s tyrosine kinase. Insulin activation of the protein kinase Akt results in phosphorylation of GSK-3 at Ser, reducing its enzymatic activity [12]. We found that G␣q(Q209L) blocks insulin and platelet-derived growth factor activation of Akt by inhibiting its upstream regulator phosphatidylinositol 3-kinase (PI3K) [20]. Active G␣q still inhibited PI3K/Akt in cells treated with U73122, a PLC inhibitor, suggesting that the inhibitory mechanism is independent of the canonical PLC pathway [20]. We show here that G␣q(Q209L) does induce the activation of GSK-3, and use of a G␣q(Q209L) mutant that cannot activate PLC indicates that this response is independent of the canonical PLC pathway. We identify Csk (C-terminal Src kinase), which has been shown to be activated by G␣q(Q209L) [21], as a tyrosine kinase that activates GSK-3
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