Phosphoinositide 3-kinase γ inhibits cardiac GSK-3 independently of Akt
Authors: Sathyamangla V Naga Prasad
John David Mosinski
Neelakantan T Vasudevan
Maradumane L Mohan
Elizabeth E Martelli
Manveen K Gupta
Babal K Jha
Journal: Science signaling (2013)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: cardiac; phosphoinositide 3-kinase α; PI3Kγ; Akt; glycogen synthase kinase-3
Affiliations: Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195, USA.

Abstract:  Activation of cardiac phosphoinositide 3-kinase α (PI3Kα) by growth factors, such as insulin, or activation of PI3Kγ downstream of heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors stimulates the activity of the kinase Akt, which phosphorylates and inhibits glycogen synthase kinase-3 (GSK-3). We found that PI3Kγ inhibited GSK-3 independently of the insulin-PI3Kα-Akt axis. Although insulin treatment activated Akt in PI3Kγ knockout mice, phosphorylation of GSK-3 was decreased compared to control mice. GSK-3 is activated when dephosphorylated by the protein phosphatase 2A (PP2A), which is activated when methylated by the PP2A methyltransferase PPMT-1. PI3Kγ knockout mice showed increased activity of PPMT-1 and PP2A and enhanced nuclear export of the GSK-3 substrate NFATc3. GSK-3 inhibits cardiac hypertrophy, and the hearts of PI3Kγ knockout mice were smaller compared to those of wild-type mice. Cardiac overexpression of a catalytically inactive PI3Kγ (PI3Kγ(inact)) transgene in PI3Kγ knockout mice reduced the activities of PPMT-1 and PP2A and increased phosphorylation of GSK-3. Furthermore, PI3Kγ knockout mice expressing the PI3Kγ(inact) transgene had larger hearts than wild-type or PI3Kγ knockout mice. Our studies show that a kinase-independent function of PI3Kγ could directly inhibit GSK-3 function by preventing the PP2A-PPMT-1 interaction and that this inhibition of GSK-3 was independent of Akt. 

Retracted: No

PubPeer comments:
 Fig 6: Inhibition of PP2A-mediated dephosphorylation GSK-3 by PI3Ku03b3 increases GSK-3u2013 regulated transcription factor and target gene expression. ![file] Cell lysates from Panel A and C are duplicated. Similar appears to be in panel B and D as well, however, an enhanced band intensities are noted in panel B. Can authors explain this as none was written in Fig 6 legend. Furthermore,the Fig 6(F) legend stated:  Cardiac lysates from WT and PI3Ku03b3 KO mice were immunoblotted with anti-ANP antibody. The blots were stripped and reprobed with antiu2013u03b2-actin antibody . Was it a different lysate or total lysate; NOT from nuclear or cytosolic, right? , 
 Although the results section and figure legend discusses this interesting observation, apparently it looks like we did not do a good job in descripting it as it was not clear to the reader. Please find below the explanation for the query. Fig 6 A and C. The input for both panel A and C are the same nuclear lysates. Panel A is immunoprecipitation (IP) of phospho-NFATc3 and panel C is IP of NFATc3. The IPs have been immunoblotted (IB) for co-immunoprecipitating GSK-3. Since input for both IPs are same, same blot is presented. The purpose is to show that GSK-3 localization is not changed, but only association with NFAT is changed in these mice. Similarly, Fig 6 B and D represents cytosolic fractions wherein the input for both panel B and D are the same cytosolic lysates. Panel B is IP for phospho-NFATc3 and panel D is IP for NFATc3. The IPs have been immunoblotted (IB) for co-immunoprecipitating GSK-3. Again the purpose is to show GSK-3 levels in the cytosol is not changed but only to show the association with NFAT. The blot in the Fig 6F is from total cardiac lysates, not from nuclear or cytosolic fractions. Hope this clarifies the query and if you have any questions, please do not hesitate to contact us. , 
 #2 Thanks for the clarification. No further question. , 
 Glad that we were able to clarify your questions. Thanks!! ,