Abstract
Glycogen synthase kinase 3 (GSK3) β and α are serine/threonine kinases involved in many biological processes. A primary mechanism of GSK3 activity regulation is phosphorylation of N-terminal serine (S) residues (S9 in GSK3β, S21 in GSK3α). Phosphorylation is inhibitory to GSK3 kinase activity because the phosphorylated N-terminus acts as a competitive inhibitor for primed substrates. Despite widespread interest in GSK3 across most fields of biology, the research community does not have reagents that specifically react with nonphosphoS9/21 GSK3β/α (the so-called “active” form). Here, we describe two novel monoclonal antibodies that specifically react with nonphosphoS9/21 GSK3β/α in multiple species (human, mouse, and rat). One of the antibodies is specific for nonphospho-S9 GSK3β (clone 12B2) and one for nonphospho-S9/21 GSK3β/α (clone 15C2). These reagents were validated for specificity and reactivity in several biochemical and immunochemical assays, and they show linear detection of nonphosphoS GSK3. Finally, these reagents provide significant advantages in studying GSK3β regulation. We used both antibodies to study the regulation of S9 phosphorylation by Akt and protein phosphatases. We used 12B2 (due to its specificity for GSK3β) and to demonstrate that protein phosphatase inhibition reduces nonphospho-S9 GSK3β levels and lowers kinase activity within cells. The ability to use the same reagent across biochemical, immunohistological and kinase activity assays provides a powerful approach for studying serine-dependent regulation of GSK3β/α.
Highlights
Glycogen synthase kinase (GSK) was described in the 80’s for its role in regulating glycogen synthase (Embi et al, 1980; Rylatt et al, 1980)
Mouse N00 was immunized with the N-terminal Keyhole Limpet hemocyanin (KLH) npS9 GSK3β peptide and animals T10 and E10 were immunized with a mixture of 3 peptides (N-term KLH, arginine enantiomer and the tandem npS9 GSK3β peptides)
Novel GSK3β Antibodies Demonstrate that Protein Phosphatases Reduce npS9 GSK3β Levels and Kinase Activity through an Akt-Independent Pathway
Summary
Glycogen synthase kinase (GSK) was described in the 80’s for its role in regulating glycogen synthase (Embi et al, 1980; Rylatt et al, 1980). GSK3 is a highly conserved serine (S)/threonine (T) kinase and two genes encode the α and β paralogs. The primed residue binds the primed substrate pocket bringing the target S/T residue into the kinase domain (Beurel et al, 2015). A number of GSK3β substrates such as glycogen synthase, β-catenin and tau are phosphorylated by GSK3β after being primed by other kinases (Zhang et al, 1993; Hagen and Vidal-Puig, 2002; Li et al, 2006), but not all GSK3β substrates require priming (Wang et al, 1994b)
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