Serine phosphorylation sites on IRS2 activated by angiotensin II and protein kinase C to induce selective insulin resistance in endothelial cells
Authors: Qian Li
Jonathon Winnay
Koji Mizutani
Edward P Feener
Katharine D&#x27;Aquino
George L King
Morris F White
Kyoungmin Park
Christian Rask-Madsen
Akira Mima
Yasutaka Maeda
Journal: Molecular and Cellular Biology (2013)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: Protein kinase C; insulin resistance; endothelial cells; phosphorylation; IRS2
Affiliations: Section of Vascular Cell Biology, Joslin Diabetes Center, Harvard Medical School, Boston, Massachusetts, USA., Section of Vascular Cell Biology, Joslin Diabetes Center, Harvard Medical School, Boston, Massachusetts, USA, Children's Hospital Boston, Boston, Massachusetts, USA

Abstract:  Protein kinase C (PKC) activation, induced by hyperglycemia and angiotensin II (AngII), inhibited insulin-induced phosphorylation of Akt/endothelial nitric oxide (eNOS) by decreasing tyrosine phosphorylation of IRS2 (p-Tyr-IRS2) in endothelial cells. PKC activation by phorbol ester (phorbol myristate acetate [PMA]) reduced insulin-induced p-Tyr-IRS2 by 46% ± 13% and, similarly, phosphorylation of Akt/eNOS. Site-specific mutational analysis showed that PMA increased serine phosphorylation at three sites on IRS2 (positions 303, 343, and 675), which affected insulin-induced tyrosine phosphorylation of IRS2 at positions 653, 671, and 911 (p-Tyr-IRS2) and p-Akt/eNOS. Specific PKCβ2 activation decreased p-Tyr-IRS2 and increased the phosphorylation of two serines (Ser303 and Ser675) on IRS2 that were confirmed in cells overexpressing single point mutants of IRS2 (S303A or S675A) containing a PKCβ2-dominant negative or selective PKCβ inhibitor. AngII induced phosphorylation only on Ser303 of IRS2 and inhibited insulin-induced p-Tyr911 of IRS2 and p-Akt/eNOS, which were blocked by an antagonist of AngII receptor I, losartan, or overexpression of single mutant S303A of IRS2. Increases in p-Ser303 and p-Ser675 and decreases in p-Tyr911 of IRS2 were observed in vessels of insulin-resistant Zucker fatty rats versus lean rats. Thus, AngII or PKCβ activation can phosphorylate Ser303 and Ser675 in IRS2 to inhibit insulin-induced p-Tyr911 and its anti-atherogenic actions (p-Akt/eNOS) in endothelial cells. 

Retracted: No

PubPeer comments:
 Could the authors please check Figures 4G and 9A? The panels look very similar to each other, but Figure 4G is supposed to show  *Immunoblot analysis for p-Tyr671 and p-Tyr911 of IRS2 in lysates from BAEC expressing SM-IRS2 (S303A) or SM-IRS2 (S675A) in absence or presence of PKCb2.* ![file] , 
 Dear Dr. Bik, Thank you for the careful reading of our MCB paper u201c Molecular and Cellular Biology (2013) - pubmed: 23775122 doi: 10.1128 mcb.00506-13 issn: 0270-7306 issn: 1098-5549u201d. As you have pointed out, Fig. 4G and Fig 9A are very similar. In fact, they are the same and the description in the figure legends described the same method. We also described them the same way in the text. We agree this is unusual since this cost more space for publication. However, this paper is unusual since it contains 13 figures and a total of 74 panels and many different sites of phosphorylation in the publications. From lab memory of 2013, we suggest this was done to facilitate the readers for not having to refer back to Fig 4G in order to see which Thr Ser were phosphorylated on IRS2 by PKCuf062ll as compared to PMA and Angiotensin II which were shown in Fig 10. We are happy to discuss with MCB to print an explanation, if necessary. Dr. George L. King , 
 Dear Dr. King, thank you for your reply. Although we both agree this is the same image published twice, the legends appear to describe different experiments, as I wrote above. Legend Figure 4G:  Immunoblot analysis for p-Tyr671 and p-Tyr911 of IRS2 in lysates from BAEC *expressing SM-IRS2 (S303A) or SM-IRS2 (S675A)* in absence or presence of *PKCb2*. Legend Figure 9A:  Immunoblot analysis for p-Tyr671 and 911 of IRS2 in lysates from BAEC *expressing PKCb2 by adenoviral infection of PKCb2* in absence or presence of *insulin.* I italicized the parts that look different. How could these be the same experiments? , 
 #3 Re: * How could these be the same experiments? * I started to offer a potential explanation, although I end up thinking the legend for 4G is inaccurate. The labeling of the Figure indicates that each Figure includes variation of two conditions: * (i) presence vs. absence of PKCb2 (labeled at top: Ad-GFP vs. Ad-PKCbII) * (ii) presence vs. absence of insulin (labeled at bottom: - vs. +) In addition, there is also variation of the SM-IRS2 variant in the Ad-PKCbII cells. But it does not seem that Fig 4G is really comparing the two SM-IRS2 variants in both the  absence or presence of PKCbII , but rather only in its presence. ,