Inhibitory Phosphorylation of Glycogen Synthase Kinase-3 (GSK-3) in Response to Lithium

Abstract

Glycogen synthase kinase-3 (GSK-3) is a critical, negative regulator of diverse signaling pathways. Lithium is a direct inhibitor of GSK-3 and has been widely used to test the putative role of GSK-3 in multiple settings. However, lithium also inhibits other targets, including inositol monophosphatase and structurally related phosphomonoesterases, and thus additional approaches are needed to attribute a given biological effect of lithium to a specific target. For example, lithium is known to increase the inhibitory N-terminal phosphorylation of GSK-3, but the target of lithium responsible for this indirect regulation has not been identified. We have characterized a short peptide derived from the GSK-3 interaction domain of Axin that potently inhibits GSK-3 activity in vitro and in mammalian cells and robustly activates Wnt-dependent transcription, mimicking lithium action. We show here, using the GSK-3 interaction domain peptide, as well as small molecule inhibitors of GSK-3, that lithium induces GSK-3 N-terminal phosphorylation through direct inhibition of GSK-3 itself. Reduction of GSK-3 protein levels, either by RNA interference or by disruption of the mouse GSK-3␤ gene, causes increased N-terminal phosphorylation of GSK-3, confirming that GSK-3 regulates its own phosphorylation status. Finally, evidence is presented that N-terminal phosphorylation of GSK-3 can be regulated by the GSK-3-dependent protein phosphatase-1⅐inhibitor-2 complex.

The protein phosphatase-1/inhibitor-2 complex differentially regulates GSK3 dephosphorylation and increases sarcoplasmic/endoplasmic reticulum calcium ATPase 2 levels

“…Initially, Zhang et al [46] reported the existence of an autoregulatory loop whereby GSK3 regulates its own phosphorylation at Ser21/9. In this case, the phosphorylation and inhibition of I-2 by GSK3 results in increased PP1 activity and decreased Ser21/9 phosphorylation of GSK3.…”

“…The regulation of Ser21/9 phosphorylation has been attributed to the actions of protein phosphatase-2A (PP2A) [40,43,27,38] and protein phosphatase-1 (PP1) [46,34,41]. The PP1 holoenzyme is comprised of a 37 kDa catalytic subunit which associates with inhibitory subunits and targeting subunits.…”

Assessment of circadian function in fibroblasts of patients with bipolar disorder

“…The observed reduced level of p-GSK3b, thus higher GSK3b activity in bipolar fibroblasts, may also underlie the rationale for lithium treatment of bipolar disorder since lithium directly inhibits GSK3b resulting in N-terminal phosphorylation of GSK3b at serine 9. 61 It was reported that the level of p-GSK3b in peripheral blood mononuclear cells from subjects with bipolar disorder under lithium treatment is significantly higher than that in healthy control subjects, 100 although no difference was detected between lithium-free bipolar and healthy controls. We postulate that the genetically heterogeneous nature of the samples may account for the failure of detecting p-GSK3b level difference between lithiumfree bipolar and healthy controls, as we encountered when including samples from members of non-Amish family 884 in our analysis.…”

“…Among these kinases, GSK3b is of particular interest here because it was reported to be one of the direct targets of lithium, 60 which is an effective therapy for bipolar disorder. 22,23 The activity of GSK3b can be inhibited by lithium, 60 which results in the phosphorylation of GSK3b at Ser9, 61 and the level of p-GSK3b indicates the activity of GSK3b. 28 Similar to the circadian pacemaker in the SCN in mammals, cultured cells, including fibroblasts, harbor self-sustained and cell-autonomous circadian clocks that persist even during cell division.…”

Glycogen synthase kinase-3β positively regulates the proliferation of human ovarian cancer cells

“…We further transfected the SKOV3 cells with GID5-6, a peptide inhibitor of GSK-3β derived from the GSK-3β interaction domain of axin [20]. The control GID5-6LP contains a single amino acid mutation rendering it unable to interact with GSK-3β [20,21]. Transfection with GID5-6 led to decrease of BrdU intake in SKOV3 cells (20% positive), compared with cells transfected with GID5-6LP (32.4% positive).…”