The Substrate Specificity of GSK3

Soon after its discovery, it was noted that GSK3 could only phosphorylate glycogen synthase efficiently if glycogen synthase had already been phosphorylated by CK2 [6]. Phosphorylation by CK2 did not inhibit glycogen synthase, but primed this enzyme for phosphorylation by GSK3. Elegant studies by Roach and his colleagues then established that the substrate specificity requirements of GSK3 are unique: the protein kinase phosphorylating serine and threo-nine residues that lie in Ser/Thr-Xaa-Xaa-Xaa-pSer/pThr, where pSer is phosphoserine, pThr is phosphothreonine, and Xaa is any amino acid [7]. In the case of glycogen synthase, the phosphorylation of Ser656 by CK2 forms the recognition site for the GSK3-catalyzed phosphorylation of Ser652. This, in turn, acts as the recognition site for the phosphorylation of Ser648 and so on, leading to the sequential phosphorylation of Ser644 and Ser640, the phosphory-lation of the last two residues having the major effect on activity [7].

GSK3 phosphorylates many proteins in vitro, some of which are likely to be physiological substrates. For example, it phosphorylates Ser535 on the e-subunit of eukaryotic protein synthesis initiation factor eIF2B [8,9], which is the guanosine triphosphate (GTP)/guanosine diphosphate (GDP)s exchange factor that converts eIF2 to its active GTP-bound form, thereby allowing it to form a ternary complex with Met-tRNA and the 40S ribosome. The phosphorylation of Ser535 inactivates eIF2B, resulting in an inhibition of protein synthesis. The phosphorylation of Ser535 by GSK3 is dependent on the prior phosphorylation of Ser539. This residue is not phosphorylated by CK2 but, at least in vitro, is phosphorylated specifically by the dual-specificity, tyrosine-phosphorylated and -regulated kinase (DYRK) [10]. However, whether a DYRK isoform phosphorylates eIF2B at Ser539 in vivo has not yet been established. GSK3 is also reported to phosphorylate ATP-citrate lyase at Thr446 and Ser450 [11,12] and the cAMP-response element binding protein (CREB) at Ser129 [13]. In these cases, phosphorylation of ATP-citrate lyase and CREB depends on the prior phosphorylation of Ser454 and Ser133, respectively, by protein kinases such as cAMP-dependent protein kinase A (PKA). Thus, it is clear that the nature of the priming kinase varies from substrate to substrate. In the case of glycogen synthase and eIF2B, the level of phosphorylation of the priming site is high, even in quiescent cells, because CK2 and DYRK are constitutively active protein kinases. However, in the case of ATP-citrate lyase and CREB, the level of phosphorylation of the priming site increases in response to several extracellular signals, such as those that elevate cAMP and activate PKA.

The site on GSK3 that binds the priming phosphate of substrates has been identified. It is located in the N-terminal lobe of the catalytic domain near the activation loop present in many protein kinases and contains three crucial basic residues (Arg96, Arg180, and Lys205 in the P-isoform) that interact directly with the priming phosphate [14-16]. Interestingly, the three-dimensional structure of GSK3 most closely resembles that of mitogen-activated protein kinase (MAPK) family members. The activation of MAPKs requires the phosphorylation of a threonine and a tyrosine residue located in a Thr-Xaa-Tyr sequence in the activation loop, which is catalyzed by dual specificity MAPK kinases (MKKs). Intriguingly, the phosphothreonine residue in the activation loop of MAPKs interacts with the same three basic residues that bind the priming phosphate in substrates of GSK3 [15]. Moreover, GSK3 is itself phosphorylated at a tyrosine residue located in a position equivalent to that of the phosphotyrosine residue in MAPKs [17]. Thus, the way in which the active form of GSK3 is generated may be analogous to that of MAPKs, except that the active conformation is induced when the priming phosphate of the substrate binds to GSK3 [15]. Unlike the MAPKs, the phosphotyrosine residue in GSK3 (Tyr279 of GSK3a, Tyr216 of GSK3P) appears to be phosphorylated constitutively in most mammalian cells [17,18]. GSK3P expressed in Escherichia coli (a bacterium thought to lack protein tyrosine kinase activity) is phosphorylated at Tyr216 and wild-type GSK3P but is not a catalytically inactive mutant and becomes phosphorylated at Tyr216 when transfected into human HEK 293 cells (Frame and Cohen, unpublished data). These observations suggest that the phosphorylation of the tyrosine residue in GSK3 is catalyzed by GSK3 itself. In contrast, there is evidence that in the slime mold Dictyostelium discoideum GSK3 is activated by tyrosine phosphorylation, which is catalyzed by the protein kinase ZAK1 [19]. However, the tyrosine residues that become phosphorylated have not yet been identified, and ZAK1 homologs do not appear to be present in the human genome. Nevertheless, the possibility that the tyrosine phosphorylation of GSK3 may be catalyzed by another protein kinase in some mammalian cells cannot be excluded, because the phosphorylation of GSK3P at Tyr216 has been reported to increase in neuronal cells after cerebral damage or after withdrawal of nerve growth factor (NGF) from the culture medium [20,21].

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