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  • Protein Phosphatase 1-α Regulates AS160 Ser588 and Thr642 Dephosphorylation in Skeletal Muscle.

Protein Phosphatase 1-α Regulates AS160 Ser588 and Thr642 Dephosphorylation in Skeletal Muscle.

Diabetes (2016-06-02)
Pragya Sharma, Edward B Arias, Gregory D Cartee
ABSTRACT

Akt substrate of 160 kDa (AS160) phosphorylation on Thr(642) and Ser(588) by Akt is essential for insulin's full effect on glucose transport. However, protein phosphorylation is determined by the balance of actions by kinases and phosphatases, and the specific phosphatase(s) controlling AS160 dephosphorylation is (are) unknown. Accordingly, we assessed roles of highly expressed skeletal muscle serine/threonine phosphatases (PP1, PP2A, PP2B, and PP2C) on AS160 dephosphorylation. Preliminary screening of candidate phosphatases used an AS160 dephosphorylation assay. Lysates from insulin-stimulated skeletal muscle were treated with pharmacological phosphatase inhibitors and assessed for AS160 Ser(588) and Thr(642) dephosphorylation. AS160 dephosphorylation on both phosphorylation sites was unaltered by PP2B or PP2C inhibitors. Okadaic acid (low dose inhibits PP2A; high dose inhibits PP1) delayed AS160 Ser(588) (both doses) and Thr(642) (high dose only) dephosphorylation concomitant with greater Akt phosphorylation (both doses). AS160 was coimmunoprecipitated with PP1-α but not with PP1-β, PP1-γ1, or PP2A. Recombinant inhibitor-2 protein (a selective PP1 inhibitor) delayed AS160 dephosphorylation on both phosphorylation sites without altering Akt phosphorylation. Furthermore, knockdown of PP1-α but not PP1-β or PP1-γ1 by small interfering RNA caused greater AS160 Ser(588) and Thr(642) phosphorylation concomitant with unaltered Akt phosphorylation. Together, these results identified PP1-α as a regulator of AS160 Thr(642) and Ser(588) dephosphorylation in skeletal muscle.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Anti-PP1γ1 Antibody, Upstate®, from rabbit
Sigma-Aldrich
Anti-AS160 Antibody, from rabbit, purified by affinity chromatography
Sigma-Aldrich
Normal Rabbit IgG, Normal Rabbit IgG Polyclonal Antibody control validated for use in Immunoprecipitation & Western Blotting.
Sigma-Aldrich
Anti-PP1β Antibody, Upstate®, from rabbit