Enhancement of p38 Mitogen-activated Protein Kinase Activity by Phosphorylated Glia Maturation Factor

Abstract

We previously demonstrated that glia maturation factor (GMF), a 17-kDa brain protein, can be phosphorylated in test tube by several protein kinases, and that endogenous GMF is rapidly phosphorylated upon stimulation of astrocytes by phorbol 12-myristate 13-acetate. We further observed that protein kinase A (PKA)-phosphorylated GMF is a potent inhibitor (IC50 = 3 nM) of the ERK1/ERK2 (p44/p42) subfamily of mitogen-activated protein (MAP) kinase. We now report that, by contrast, PKA-phosphorylated GMF strongly enhances the activity of a related but distinct subfamily of MAP kinase, the p38 MAP kinase, showing an increase of 60-fold over baseline and an EC50 of 7 nM. Non-phosphorylated GMF or GMF phosphorylated by other kinases exhibits only minimal effect. The intracellular interaction of PKA, GMF, and p38 is supported by the phosphorylation of GMF upon cellular stimulation by forskolin (blocked by PKA inhibitor) and by the co-immunoprecipitation of p38 with GMF from cell lysates. Withdrawal of nerve growth factor from PC12 leads to increased GMF phosphorylation with a time course similar to that reported for p38 activation. The results correlate well with a previous report that ERK and p38 carry out opposing functions and implicate GMF as a regulator of major cellular events.

Protein Kinase A (PKA)- and Protein Kinase C-phosphorylated Glia Maturation Factor Promotes the Catalytic Activity of PKA

“…4 shows that there were at least three defined protein bands capable of phosphorylating GMF, two of which were identical in electrophoretic mobility with PKA and PKC, whereas a third band was unidentified. Although the results may not necessarily reflect the in vivo situation, when taken together with our earlier findings that phorbol ester (activator of PKC) and forskolin (activator of PKA) stimulate the phosphorylation of endogenous GMF in live cells (4,6), a strong argument can be made for the activation of endogenous GMF by endogenous PKC and PKA.…”

“…Thus, it is possible that various phosphorylated isoforms of GMF may be generated inside the cell by kinases differentially stimulated by external stimuli. Although no isoform of GMF, phosphorylated or nonphosphorylated, possesses kinase or phosphatase activity (4,5), we have shown recently that PKAphosphorylated GMF is a potent inhibitor of ERK (5) and also an enhancer of p38 (6); both are subfamilies of MAP kinase, suggesting that GMF is a bifunctional regulator of the MAP kinase cascades. To find out any additional locations where GMF can regulate, we have tested the effect of GMF and GMF-P on PKA.…”

Protection of glioblastoma cells from cisplatin cytotoxicity via protein kinase Cι-mediated attenuation of p38 MAP kinase signaling

“…Microarray analysis showed that PKCi depletion increased the levels of glia maturation factor b (GMFb), a protein which has previously shown to be an enhancer of p38 MAP kinase signaling ( Figure 5B) (Lim and Zaheer, 1996). This suggested that PKCi could protect cells from apoptosis by preventing GMFb-mediated enhancement of p38 MAP kinase signaling.…”

“…This beneficial effect comes at a price, as GMFb sensitizes both astrocytes and proximal tubular kidney cells to oxidative injury (Kaimori et al, 2003;Zaheer et al, 2004). At least part of this sensitization is due to GMFb's ability to enhance p38 MAP kinase activity (Kaimori et al, 2003;Zaheer et al, 2004), which appears to involve a physical association between GMFb and this kinase in cells (Lim and Zaheer, 1996).…”

“…This repression of GMFb gene expression by PKCi may occur either by a direct mechanism, or indirectly as a consequence of the regulation of other genes. As GMFb has been shown to enhance p38 MAP kinase signaling (Lim and Zaheer, 1996), it was predicted that the increase in GMFb levels upon PKCi depletion would lead to an enhanced p38 MAP kinase activation in response to cisplatin. This was the case, as cisplatin-activated p38 MAP kinase to a greater extent in cells depleted of PKCi.…”