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Interactive Pathway: GSK-3 Technical Overview | | | | | Glycogen synthase kinase-3 (GSK-3): Technical Overview
Glycogen synthase kinase-3 (GSK-3), a multifunctional serine/threonine kinase, is a key regulator of numerous signaling pathways. Two isoforms of GSK-3 are reported in mammals: a 51 kDa GSK-3a and a 47 kDa GSK-3b. These two isoforms exhibit about 98% homology in their kinase domains, but share only about 36% identity in the last 76 C-terminal amino acid residues. GSK-3a contains a glycine-rich extension at its N-terminus. A minor (~15% of total) splice variant of GSK-3b, GSK-3b2, has also been identified, which contains a 13-residue insert within the kinase domain. It exhibits reduced kinase activity towards tau protein compared with ‘unspliced’ GSK-3b. GSK-3b2 is localized primarily to neuronal cell bodies, unlike unspliced GSK-3b that is also found in neuronal processes.
GSK-3 is normally active in cells and is regulated through inhibition of its activity. GSK-3 shows a preference for target proteins that are pre-phosphorylated at a “priming” residue located C-terminal to the site of GSK-3 phosphorylation. Priming phosphorylation, although not absolutely required, enhances the efficiency of phosphorylation of most GSK-3 substrates. Phosphorylation of a Thr residue in the activation loop (T-loop) is considered to be essential for several protein kinases, such as Cdk2, p38g, and ERK2 that are closely related to GSK-3. This phosphorylation at Thr is also required by p38g and ERK2 to open up the catalytic site for substrate access. The T-loop of GSK-3a is phosphorylated at Tyr279 and GSK-3b at Tyr216, which play a role in forcing open the substrate-binding site of the enzyme. Uniquely, the T-loop of GSK-3 does not undergo any Thr phosphorylation. The function of missing pThr in the T-loop of GSK-3 is carried out by the phosphorylated residue of a primed substrate that binds to a positively charged pocket consisting of Arg96, Arg180, and Lys205 (for GSK-3b). This arrangement optimizes the orientation of the kinase domain and places the substrate at the proper position within the catalytic groove for phosphorylation to take place.
GSK-3b is constitutively active in resting cells and treatment of cells with an agent, such as insulin, is shown to cause GSK-3 inactivation through a PI 3-kinase (PI 3-K)-dependent mechanism. PI 3-K-induced activation of PKB/Akt results in phosphorylation of Ser21 on GSK-3a and Ser9 on GSK-3b, which inhibits GSK-3 activity. The phosphorylated N-terminus becomes a primed pseudosubstrate that occupies the positive binding pocket and the active site of the enzyme, and acts as a competitive inhibitor for true substrates. This prevents phosphorylation of substrates. Arg96 is shown to be a crucial component of the positive pocket that binds primed substrates. An Arg96 to Ala96 mutation disrupts the pocket in a way that primed substrates can no longer bind and, hence, the enzyme remains active. Also, the Ser9-phosphorylated pseudosubstrate is no longer capable of inactivating the enzyme. Small molecule inhibitors that fit in the positively charged pocket of the kinase domain of GSK-3b are useful for selectively inhibiting primed substrates. Several known GSK-3 substrates participate in a wide spectrum of cellular processes, including glycogen metabolism, transcription, translation, cytoskeletal regulation, intracellular vesicular transport, cell cycle progression, and apoptosis. Phosphorylation of these substrates by GSK-3b usually has an inhibitory effect.
GSK-3b plays a key inhibitory role in the Wnt signaling pathway. Wnt genes encode a large family of secreted, cysteine-rich proteins that are important in development and in maintenance of adult tissues. Abnormalities in Wnt signaling are reported to promote both human degenerative diseases and cancer. Several groups have shown that b-catenin is a primed substrate for GSK-3b, with casein kinase I (CKI) acting as the priming kinase. In this capacity, CKI functions as a negative regulator of Wnt signaling since it promotes GSK-3 function. In unstimulated cells, CKI phosphorylates b-catenin on Ser45, priming it for further phosphorylation on Ser41, 35, and 33 by GSK-3b in a sequential manner, thereby allowing b-catenin to be ubiquitinated for proteasomal degradation. It has been suggested that the ankyrin repeat protein, Diversin, may help recruit CKI to the destruction complex. Wnt stimulation activates the receptor Frizzled, which then signals through Dishevelled (Dvl) to inactivate b-catenin phosphorylation. Unphosphorylated b-catenin translocates to the nucleus where it transactivates genes regulated by TCF/LEF transcription factors. Another key player in the regulation of the Wnt signaling pathway is GBP/FRAT, a GSK-3-binding protein. Binding of GBP/FRAT to GSK- 3b prevents GSK-3b from binding to axin and thus it interferes with b-catenin phosphorylation. GBP/FRAT also plays a significant role in the nuclear export of GSK- 3b. This suggests that GBP/FRAT may be involved in regulating the access of GSK-3 to substrates partitioned between the nucleus and the cytoplasm. Any mutation that prevents the binding of GSK-3b to GBP/FRAT causes nuclear localization of GSK-3b. A small peptide derived from FRAT, FRATtide, is reported to prevent axin-GSK-3 interaction and block the phosphorylation of both axin and b-catenin. In the Wnt signaling pathway GSK-3b appears to be insulated from regulators of GSK-3b that lie outside of the Wnt pathway. Insulin signaling that leads to inhibition of GSK-3 via phosphorylation at either Ser9 or Ser21 does not cause accumulation of b-catenin. Mutations in b-catenin that prevent its phosphorylation by GSK-3b are common in skin, colon, prostate, liver, endometrial, and ovarian cancers.
GSK-3b also plays a significant role in Hedgehog signaling. Sonic Hedgehog (Shh) has been implicated in several embryonic developmental processes and it displays inductive, proliferative, neurotrophic, and neuroprotective properties. Response to Shh signaling is controlled by two transmembrane proteins, Patched (Ptc), a twelve-span transmembrane protein, and Smoothened (Smo), a seven transmembrane receptor protein. Ptc acts as an inhibitor of Smo activation. Binding of Shh to Ptc lifts the inhibitory effect on Smo, leading to the activation of the Shh signaling cascade. Wnt and Shh often work in concert to set the embryonic development pattern. The Wnt pathway uses b-catenin to transduce its signals to the nucleus, whereas the Shh pathway employs a 155 amino acid protein, Cubitus interruptus (Ci155) in Drosophila or Gli in mammals. In vertebrates three Gli proteins (Gli1, Gli2, and Gli3) have been reported, Gli1 and Gli2 function primarily as activators, and Gli3 has a repressor role. In the absence of any Shh signal, Ci is targeted for proteolysis, generating a truncated 75-residue amino acid form (Ci75), which functions as a transcriptional repressor. GSK-3b, in combination with CKI and the priming PKA, phosphorylates Ci155 (and probably Gli) and targets it for proteolytic processing in the absence of a Shh signal. Activation of Shh signaling results in translocation of Ci155/Gli to the nucleus, where it activates Hh target genes. Although GSK-3b phosphorylation of the mammalian homologs of Ci have yet to be reported, they all contain multiple GSK-3 consensus sites next to PKA sites. Suppressor of Fused (SuFu) interacts directly with Gli proteins, repressing Shh signaling while Dyrk1 acts by a distinct pathway to stimulate Gli1 activation of transcription. Over-signaling by Shh appears to be involved in the initiation and propagation of some tumors of the muscle, skin, and nervous system.
Abnormalities in pathways that use GSK-3 as a regulator have been linked to several disease conditions. Hence, GSK-3 has emerged as a potential therapeutic target, particularly in non-insulin-dependent diabetes mellitus, Alzheimer’s disease, developmental disorders, and cancer. Several new GSK-3 inhibitors have recently been developed, most of which act in an ATP competitive manner. Inhibitors belonging to aloisines, the paullones, and the maleimide families have shown promise as therapeutic agents. Due to its involvement in multiple pathways, selectivity of GSK-3 inhibition is an important factor in the development of inhibitors for therapeutic applications.
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