This proposal investigates a novel hypertrophic and anti-apoptotic pathway that is mediated by Ras GTPase-activating protein (RasGAP). Our previous results show that nGAP plays a role in cardiac cell hypertrophy by increasing RNA polymerase II (RNA pol II) activity and RNA and protein synthesis through as of yet unknown mechanisms. While investigating this we discovered that RasGAP interacts with Akt and the muscle isoform of the actin-binding protein filamin (FLN2). This proposal will explore the role of these interactions in mediating RasGAP's hypertrophic effects. Our preliminary results show the following: 1. RasGAP binds Akt and filamin simultaneously, through its SH2 and SH3 domains, respectively. 2. RasGAP and filamin concentrations and Akt activity are significantly increased during pressure overload cardiac hypertrophy. 3. When the increase in RasGAP protein is reproduced in tissue culture or a cardiac specific transgenic mouse model, Akt activity and nuclear translocation are enhanced. 4. RasGAP's interaction with the PH domain of Akt mediates its activation via integrin-linked kinase (ILK), which phosphorylates Akt on Ser-473.5. ILK is also upregulated during cardiac hypertrophy. 6. Through activation of Akt, RasGAP is rendered anti-apoptotic, a function that is also dependent on its association with filamin. 7. And most interestingly, Akt mediates RNA pol II phosphorylation. Accordingly our hypothesis is that an increase in free RasGAP concentration during cardiac hypertrophy promotes the formation of a RasGAP/FLN2/Akt complex, which may be further augmented by an increase in FLN2 protein. FLN2, which binds beta-integrins, will recruit this complex to the proximity of ILK (which also binds beta-integrins) and, in turn, will result in phosphorylating Akt on Ser-473. This modification will induce chronic activation and nuclear translocation of Akt, which in turn will mediate mRNA and protein synthesis and protect against apoptosis.
Our aims are: 1. To investigate the mechanism of RasGAP/FLN2-mediated phosphorylation and activation of Akt, 2. To investigate the role of Akt and FLN2 in mediating RasGAP-induced RNA synthesis, protein synthesis, and cell survival. 3. To determine the effect of upregulation of RasGAP and ILK on Akt and related growth and survival pathways in vivo in cardiac specific transgenic mouse models. ? ?
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