Cross-talk between the Two Divergent Insulin Signaling Pathways Is Revealed by the Protein Kinase B (Akt)-mediated Phosphorylation of Adapter Protein APS on Serine 588

Kostas D Katsanakis,Tahir S Pillay

doi:10.1074/jbc.m505959200

Kostas D Katsanakis, Tahir S Pillay

Open Access

https://doi.org/10.1074/jbc.m505959200

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Abstract

The APS adapter protein is recruited to the autophosphorylated kinase domain of the insulin receptor and initiates the phosphatidylinositol 3-kinase (PI3K)-independent pathway of insulin-stimulated glucose transport by recruiting CAP and c-Cbl. In this study, we have identified APS as a novel substrate for protein kinase B/Akt using an antibody that exhibits insulin-dependent immunoreactivity with a phosphospecific antibody raised against the protein kinase B substrate consensus sequence RXRXX(pS/pT) and a phosphospecific antibody that recognizes serine 21/9 of glycogen synthase kinase-3alpha/beta. This phosphorylation of APS is observed in both 3T3-L1 adipocytes and transfected cells. The insulin-stimulated serine phosphorylation of APS was inhibited by a PI3-kinase inhibitor, LY290004, a specific protein kinase B (PKB) inhibitor, deguelin, and knockdown of Akt. Serine 588 of APS is contained in a protein kinase B consensus sequence for phosphorylation conserved in APS across multiple species but not found in other members of this family, including SH2-B and Lnk. Mutation of serine 588 to alanine abolished the insulin-stimulated serine phosphorylation of APS and prevented the localization of APS to membrane ruffles. A glutathione S-transferase fusion protein containing amino acids 534-621 of APS was phosphorylated by purified PKB in vitro, and mutation of serine 588 abolished the PKB-mediated phosphorylation of APS in vitro. Taken together, this study identifies APS as a novel physiological substrate for PKB and the first serine phosphorylation site on APS. These data therefore reveal the molecular cross-talk between the insulin-activated PI3-kinase-dependent and -independent pathways previously thought to be distinct and divergent.

Highlights

Insulin Stimulates APS Phosphorylation on a Site Recognized by the Anti-PKBsubstrate Antibody in 3T3-L1 Adipocytes and Chinese hamster ovary (CHO) Cells—Initially, we determined whether there was evidence for protein kinase B (PKB)-mediated phosphorylation of APS in differentiated 3T3-L1 adipocytes (Fig. 1A), a widely used model for studies of insulin action and glucose transport
The results demonstrate that APS in 3T3-L1 adipocytes is captured by the immobilized anti-PKBsubstrate, indicating that APS is a physiological substrate for PKB in native untransfected cells such as 3T3-L1 adipocytes (Fig. 1A)
In this study we have identified a substrate for protein kinase B/Akt, the APS adapter protein

Summary

EXPERIMENTAL PROCEDURES

Materials—All biochemicals were purchased from Sigma except where stated. Purified, activated PKB␣/Akt was purchased from Upstate Biotechnology. The CHO.T-APS and CHO.T-APS-CAP cell lines were as previously described. 3T3-L1 adipocytes were cultured as previously described [3, 14]. The rabbit anti-PKBsubstrate and anti-phospho-GSK3 antibodies were purchased from Cell Signaling Technology. Immunoblotting was performed as previously described [14]. Site-directed Mutagenesis—Serine 588 of the rat APS cDNA was mutated to alanine using recombinant circle PCR with the following oligonucleotides (rat APS sequence): sense, 5Ј-tgcgcgcagtagatctaacgccacagagcacctg-3Ј, and antisense, 5Ј-caggtgctctgtggcgttagatctactgcgcgca-3Ј, to introduce an alanine in place of serine 588 and a unique.

Phosphorylation of APS by Protein Kinase B

RESULTS

DISCUSSION