Glycogen Synthase Kinase-3alpha Research Articles

Glycogen synthase kinase-3 beta is a dual specificity kinase differentially regulated by tyrosine and serine/threonine phosphorylation.

The enzyme glycogen synthase kinase-3 (GSK-3) has been implicated in the control of several metabolic enzymes and transcription factors in response to extracellular signals. In the past, the enzyme has been considered to be a protein Ser/Thr kinase although it was recently reported to contain Tyr(P) (Hughes, K., Nikolakaki, E., Plyte, S. E., Totty, N. F., and Woodgett, J. R. (1993) EMBO J. 12, 803-808). A cDNA encoding rabbit skeletal muscle GSK-3 beta was cloned and expressed in Escherichia coli as an active protein kinase, with apparent M(r) 46,000, capable of phosphorylating several known GSK-3 substrates. Recombinant GSK-3 beta autophosphorylated on Ser, Thr, and Tyr residues although the enzyme already contained Tyr(P) as judged by its recognition by anti-Tyr(P) antibodies. The net result of the autophosphorylation was a 3-5-fold reduction in enzyme activity. GSK-3 alpha, purified from rabbit muscle, also underwent autophosphorylation but only on Ser and Thr residues. In this case, the autophosphorylation stabilized the enzyme activity compared with the control lacking ATP/Mg2+. Of several phosphatases tested, the lambda-phage phosphatase was the most effective in dephosphorylating at Ser and Thr residues but did not dephosphorylate at Tyr residues. The action of the lambda-phosphatase caused a reactivation of GSK-3 beta to approximately 80% of the starting activity. The protein tyrosine phosphatase PTP1B was able to dephosphorylate at Tyr residues leading to a reduction in enzyme activity. A truncated form of GSK-3 beta, apparent M(r) 40,000, had a significantly higher specific activity, was defective in autophosphorylation, and was not inactivated in the autophosphorylation reaction. We conclude that GSK-3 beta is a dual specificity protein kinase in the same sense as the mitogen-activated protein kinase/ERK family of enzymes. Phosphorylation at different residues differentially controls enzyme activity, Ser/Thr phosphorylation causing inactivation and Tyr phosphorylation resulting in increased activity.

The α‐isoform of glycogen synthase kinase‐3 from rabbit skeletal muscle is inactivated by p70 S6 kinase or MAP kinase‐activated protein kinase‐1 in vitro

The alpha-isoform of glycogen synthase kinase-3 (GSK3 alpha) was inactivated by 80% towards a synthetic peptide substrate upon incubation with Mg-ATP and either MAP kinase-activated protein (MAPKAP) kinase-1 or p70 S6 kinase. Inactivation by either kinase resulted from the phosphorylation of Ser-21 and was reversed by treatment with protein phosphatase 2A1. Phosphorylation also decreased GSK3 alpha activity towards glycogen synthase, inhibitor-2 and c-jun. The specificity of GSK3 alpha was similar to GSK3 beta, but with the synthetic peptide substrate heparin stimulated the dephosphorylated form of GSK3 alpha (6-fold) more than GSK3 beta (1.8-fold). After phosphorylation, both isoforms were stimulated 15-20-fold by heparin.

Isoform differences in substrate recognition by glycogen synthase kinases 3.alpha. and 3.beta. in the phosphorylation of phosphatase inhibitor 2

Phosphorylation of inhibitor 2, the regulatory subunit of the ATP-Mg-dependent protein phosphatase, by glycogen synthase kinase 3 (GSK-3) causes activation of the phosphatase. Prior phosphorylation by casein kinase II has been shown to enhance both phosphorylation and activation of the phosphatase by GSK-3 (DePaoli-Roach, A. A. (1984) J. Biol. Chem. 259, 12144-12152). Reported here is a comparison of the phosphorylation of inhibitor 2 by two defined isoforms of GSK-3, GSK-3 alpha and GSK-3 beta. GSK-3 beta was a significantly better inhibitor 2 kinase than was GSK-3 alpha. The Vmax/Km value for GSK-3 beta was approximately 10-fold higher than that for GSK-3 alpha. GSK-3 beta phosphorylated inhibitor 2 to a stoichiometry of approximately 1.0 mol of phosphate/mol of inhibitor 2. The phosphorylation by GSK-3 beta was determined to be exclusively at Thr-72 on the basis of the inability of the enzyme to modify a mutant inhibitor 2 in which Thr-72 was changed to alanine. Prior phosphorylation by casein kinase II promoted the action of GSK-3 alpha in keeping with earlier reports using undefined GSK-3 preparations. Phosphorylation by GSK-3 beta, in contrast, was unaffected by the previous action of casein kinase II. These results suggest that there can be important differences in substrate recognition by different isoforms of the same protein kinase and may help explain why some reported GSK-3 substrates require prior phosphorylation whereas other do not.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of the glycogen synthase kinase-3 family by tyrosine phosphorylation.

Glycogen synthase kinase-3 (GSK-3) is a protein serine kinase implicated in the cellular response to insulin. The enzyme is the mammalian homologue of the zeste-white3 (shaggy) homeotic gene of Drosophila melanogaster and has been implicated in the regulation of the c-Jun/AP-1 transcription factor. In mammals this protein serine kinase is encoded by two related genes termed GSK-3 alpha and beta. Here, we demonstrate that these two proteins and the fruit fly protein are phosphorylated on tyrosine in vivo. Moreover, GSK-3 beta activity and function are shown to be dependent on tyrosine phosphorylation. The modified tyrosine residue is conserved in all members of the GSK-3 family and is equivalent to that required for activity by mitogen-activated protein (MAP) kinases. However, unlike MAP kinases, GSK-3 is highly phosphorylated on tyrosine and thus active in resting cells.

Differential regulation of glycogen synthase kinase-3 beta by protein kinase C isotypes.

In cells, stimulation of protein kinase C (PKC) results in the dephosphorylation of specific residues proximal to the DNA binding domain of c-Jun, a major component of the AP-1 transcription factor. Since phosphorylation of this region of c-Jun inhibits interaction with DNA, this pathway may contribute to PKC activation of AP-1. To determine the mechanism(s) underlying this pathway, possible interactions between PKC and proteins implicated in c-Jun regulation are being investigated. Here it is shown that glycogen synthase kinase-3 beta (GSK-3 beta), a serine/threonine kinase that specifically targets the inhibitory c-Jun phosphorylation sites, is phosphorylated in vitro by particular forms of PKC (alpha, beta 1, gamma greater than beta 2; not epsilon). By contrast, the related GSK-3 alpha is not a substrate for any of these PKC isotypes. Phosphorylation of GSK-3 beta by PKC results in its specific inactivation. These results are consistent with a model in which activation of PKC stimulates c-Jun DNA binding by inhibiting its phosphorylation by GSK-3 beta.

Baculovirus-mediated expression and characterisation of rat glycogen synthase kinase-3 beta, the mammalian homologue of the Drosophila melanogaster zeste-white 3sgg homeotic gene product.

Molecular cloning of glycogen synthase kinase-3 (GSK-3) has demonstrated the existence of a novel form, termed GSK-3 beta, which is highly related to the well characterised GSK-3 alpha protein but derived from a distinct gene. The cDNA cloning also revealed a striking degree of amino acid identity between the two GSK-3 proteins, particularly the beta-form, and the zeste-white3/shaggy (zw3sgg) homeotic gene of Drosophila melanogaster. Abrogation of zw3sgg causes pleiotropic effects on fruitfly development affecting segmental organisation and cell fate determination. In view of the potential importance of GSK-3 beta in mammalian development and the lack of previous characterisation, we have expressed this protein in insect cells using recombinant baculovirus. A rapid purification scheme has been developed yielding essentially pure GSK-3 beta protein in three chromatographic steps. The protein has autonomous protein kinase activity and similar, but not identical, substrate preferences to GSK-3 alpha. Both GSK-3 proteins activate the MgATP-dependent form of protein phosphatase-1 and thus display 'factor A' activity. Since GSK-3 beta exhibits an identical site specificity to GSK-3 alpha with respect to phosphorylation of the proto-oncogene/transcription factors c-jun and c-myc, it is likely that the Drosophila zw3sgg protein kinase has a similar specificity for such transcription factors which may underlie the pleiotropic phenotypes observed when the Drosophila homologue is mutationally inactivated.

Molecular cloning and expression of glycogen synthase kinase-3/factor A.

Glycogen synthase kinase-3 (GSK-3) is a protein-serine kinase implicated in the hormonal control of several regulatory proteins including glycogen synthase and the transcription factor c-jun. Two classes of rat brain cDNA for this enzyme have been isolated termed GSK-3 alpha and GSK-3 beta. The alpha-type encodes a 51 kd polypeptide, the sequence of which includes all of the tryptic peptides determined by protein sequence analysis of purified skeletal muscle GSK-3. The novel beta-type cDNA has the potential to encode a 47 kd protein with 85% amino acid identity to GSK-3 alpha. The two types of cDNA are the products of distinct genes as determined by genomic organization and nucleic acid sequence analysis. Both alpha and beta clones exhibit kinase activity when expressed in COS-1 cells and type-specific antibodies to GSK-3 alpha and beta detect proteins of 51 and 47 kd, respectively, in a variety of rat tissue extracts, with highest levels of both in brain. Partial purification of GSK-3 activity from bovine brain results in the isolation of active alpha and beta proteins. The physiological importance of these two proteins in cellular signal transduction is discussed.