Novel Non-phosphorylated Serine 9/21 GSK3β/α Antibodies: Expanding the Tools for Studying GSK3 Regulation

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.

Defined Tau Phosphospecies Differentially Inhibit Fast Axonal Transport Through Activation of Two Independent Signaling Pathways

“…Immunoblots were developed using a well-characterized antibody that selectively recognizes active GSK3β species dephosphorylated at the regulatory serine 9 residue (anti-dpS9-GSK3β; Figure 4 ). Dephosphorylation of this critical regulatory residue in GSK3β by any of several protein phosphatases, including PP1, represents a major activation mechanism for this kinase (Grabinski and Kanaan, 2016 ). A phosphorylation-independent antibody against GSK3β provided an internal protein loading control (anti-Total GSK3β).…”

“…The following antibodies were used for Western blots: KHC (H2 clone; Pfister et al, 1989 ); Total GSK3 (Cell Signaling Technology #D75D3); pJNK (Cell Signaling Technology #9251). Anti-dp-ser9/21-GSK3α/β (clone 15C2; Grabinski and Kanaan, 2016 ) and anti-tau (clone TNT1) were provided by Dr. Nicholas Kanaan (Michigan State University).…”

In vivo regulation of glycogen synthase kinase 3β activity in neurons and brains

“…However, it has also been noted that Ser9 phosphorylation is not an accurate indicator of GSK3β activity, and a method for properly measuring GSK3β activity in vivo has long been expected. Recently, the antibodies recognizing non-phosphorylated Ser9 were reported 36 . Here, we developed a method for estimating the kinase activity of GSK3β using Phos-tag SDS-PAGE.…”

Regulation of motor proteins, axonal transport deficits and adult-onset neurodegenerative diseases

“…Extending these findings, other kinases were found to regulate AT indirectly by modulating localized GSK3 activation (Morfini et al, 2004; Ratner et al, 1998). Unlike most protein kinases, GSK3 activation requires dephosposphorylation of an inhibitory phosphorylation site (Grabinski and Kanaan, 2016). Interestingly, it was found that inhibition of the major protein kinase CDK5 promotes activation of axonal protein phosphatase 1 (PP1), which in turn dephosphorylated and activated GSK3 (Morfini et al, 2004; Ratner et al, 1998).…”