SRY‐box 9 (SOX9): A Novel Binding Partner of Cardiac Muscle‐specific A‐Kinase Anchoring Protein (mAKAP) Signalosome

Abstract

BackgroundBy definition, A‐kinase anchoring proteins (AKAPs) bind to protein kinase A and various other signaling proteins at specific subcellular compartments to regulate downstream signal transduction. Heart specific muscle AKAP (mAKAP) is present on the nuclear envelope of cardiomyocytes and regulates cardiac hypertrophy by forming a signalosome of its binding partners. It was previously reported that hypoxia‐inducible factor 1 alpha (HIF‐1α) physically binds to mAKAP. Also, it was previously published that HIF‐1α physically interacts with SRY‐box 9 (SOX9). Chromatin immunoprecipitation (ChIP) studies showed that HIF‐1α binds to SOX9 promoter region. Interestingly, both mAKAP and SOX9 have been demonstrated to play significant role in cardiac differentiation and endocardial cushion formation, respectively. Hence, we were interested to know whether SOX9 is a part of mAKAP scaffold on the nuclear envelope.ObjectiveThe aim of the current work is to confirm that SOX9 unambiguously interacts with mAKAP; either directly or indirectly.MethodsAs HEK 293T cells express SOX9 endogenously and not mAKAP, we chose these cells for immunoprecipitation (IP) studies. Myc‐DDK tagged human mAKAP cDNA was purchased from Origene and overexpressed in HEK 293T cells using Lipofectamine 2000, according manufacturer's instructions. IP was performed using M2 affinity gels (Sigma) and immunoblotting was performed using mAKAP and SOX9 antibodies. HepG2 cell lysate was used as positive control for SOX9 expression. In another set of experiment, a Coomassie Blue stained gel was used for in‐gel digestion to perform MALDI‐TOF Mass Spectrometry, followed by data analysis.Summary of ResultsIP samples of mAKAP transfected HEK cells displayed SOX9 expression; whereas, untransfected cells did not show expression. mAKAP expression was used as a loading control and remained unchanged in all samples. Mass Spectrometric data validated our findings by demonstrating the expected mass peak for SOX9 in the IP samples.Conclusion and Future ImplicationsSOX9 was identified as a novel binding partner of mAKAP. As mAKAP and SOX9 already have a role in cardiac developmental biology, we suggest that mAKAP‐SOX9 interaction has substantial role in cardiac physiology and pathophysiology. The significance of this mAKAP‐SOX9 interaction is a part of our ongoing investigations.Support or Funding InformationR15HL124458 Scaffolding Protein Interaction Network Regulates Cardiac Cell Signaling