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Mol Cell Biol. 2002 Apr;22(7):2099-110.

Convergence of multiple signaling cascades at glycogen synthase kinase 3: Edg receptor-mediated phosphorylation and inactivation by lysophosphatidic acid through a protein kinase C-dependent intracellular pathway.

Fang X, Yu S, Tanyi JL, Lu Y, Woodgett JR, Mills GB.

Source

Department of Molecular Therapeutics, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.

Abstract

Lysophosphatidic acid (LPA) is a natural phospholipid with multiple biological functions. We show here that LPA induces phosphorylation and inactivation of glycogen synthase kinase 3 (GSK-3), a multifunctional serine/threonine kinase. The effect of LPA can be reconstituted by expression of Edg-4 or Edg-7 in cells lacking LPA responses. Compared to insulin, LPA stimulates only modest phosphatidylinositol 3-kinase (PI3K)-dependent activation of protein kinase B (PKB/Akt) that does not correlate with the magnitude of GSK-3 phosphorylation induced by LPA. PI3K inhibitors block insulin- but not LPA-induced GSK-3 phosphorylation. In contrast, the effect of LPA, but not that of insulin or platelet-derived growth factor (PDGF), is sensitive to protein kinase C (PKC) inhibitors. Downregulation of endogenous PKC activity selectively reduces LPA-mediated GSK-3 phosphorylation. Furthermore, several PKC isotypes phosphorylate GSK-3 in vitro and in vivo. To confirm a specific role for PKC in regulation of GSK-3, we further studied signaling properties of PDGF receptor beta subunit (PDGFRbeta) in HEK293 cells lacking endogenous PDGF receptors. In clones expressing a PDGFRbeta mutant wherein the residues that couple to PI3K and other signaling functions are mutated with the link to phospholipase Cgamma (PLCgamma) left intact, PDGF is fully capable of stimulating GSK-3 phosphorylation. The process is sensitive to PKC inhibitors in contrast to the response through the wild-type PDGFRbeta. Therefore, growth factors, such as PDGF, which control GSK-3 mainly through the PI3K-PKB/Akt module, possess the ability to regulate GSK-3 through an alternative, redundant PLCgamma-PKC pathway. LPA and potentially other natural ligands primarily utilize a PKC-dependent pathway to modulate GSK-3.

PMID:: 11884598; [PubMed - indexed for MEDLINE]
PMCID:: PMC133668

Free PMC Article

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FIG. 7.

Specific PKC isotypes can phosphorylate GSK-3α at serine 21 and GSK-3β at serine 9 in vitro and in vivo. (A) Different isoforms of PKC were incubated with purified GSK-3 in an in vitro kinase assay as described in Materials and Methods. Reaction mixes were diluted in SDS sample buffer and analyzed for phosphorylation of GSK-3α at serine 21 and of GSK-3β at serine 9 by immunoblotting with a GSK-3 phospho-specific antibody as described in Fig. 1. Reprobing with a phosphorylation-independent antibody against total GSK-3α and GSK-3β (substrates) was included to show equal loading among samples. The bottom panel shows activity of the PKC isotypes toward standard PKC substrates (histone H1 for classical forms and PKCɛ substrate peptide for novel and atypical forms). (B) PKCα, PKCγ, PKCη, and PKCδ were incubated with GSK-3 in the presence of the PKC inhibitor GF109203X (+GF, 0.15 μM for PKCα and PKCγ, 0.6 μM for PKCη and PKCδ) or without lipid activators (−lipid) as indicated. The reaction mixes were analyzed as in panel A. (C) HEK293 cells were cotransfected with HA-GSK-3β, along with an empty vector or a constitutively active PKC construct as indicated. Two days after transfection, cells were starved for 12 h, and lysates were prepared and analyzed for HA-GSK-3β phosphorylation levels with a GSK-3β phospho-specific antibody. Expression of transfected HA-GSK-3β was detected by immunoblotting with an anti-HA monoclonal antibody. Expression of PKC proteins was confirmed by immunoblotting with PKC isotype-specific antibodies. The anti-PKCβ1 antibody cross-reacts with PKCα, whereas the other anti-PKC antibodies did not exhibit cross-reactivity. Each of these experiments was performed at least twice with consistent results.

Convergence of Multiple Signaling Cascades at Glycogen Synthase Kinase 3: Edg Receptor-Mediated Phosphorylation and Inactivation by Lysophosphatidic Acid through a Protein Kinase C-Dependent Intracellular Pathway

Mol Cell Biol. 2002 April;22(7):2099-2110.