Intracellular Localization and Functional Effects of P ²¹ -Activated Kinase-1 (Pak1) in Cardiac Myocytes

Abstract

Abstract-We investigated intracellular localization and substrate specificity of P 21 -activated kinase-1 (Pak1) in rat cardiac myocytes. Pak1 is a serine/threonine protein kinase that is activated by Rac1/Cdc42 and important in signaling of stress responses. Yet the localization and in vivo function of Pak1 in heart cells is poorly understood. Studies reported here indicate that Pak1 physically interacts with protein phosphatase 2a and localizes to the Z-disk, cell membrane, intercalated disc, and nuclear membrane of adult rat heart myocytes. We compared levels of phosphorylation of cardiac troponin I (cTnI) in control myocytes with phosphorylation of cTnI and myosin binding protein C (C-protein) in myocytes with increased Pak1 activity. The increase in activity was induced by infection of myocytes with a recombinant adenovirus (AdPak1) containing cDNA for a constitutively active Pak1. Control cells were infected with a virus (AdLacZ) containing LacZ. Basal levels of phosphorylation of cTnI and C-protein were relatively high in the myocytes infected with AdLacZ. However, phosphorylation of cTnI and C-protein in cells expressing constitutively active Pak1 was significantly reduced compared with those expressing LacZ. Measurement of Ca 2ϩ tension relations in single myocytes demonstrated that this reduction in phosphorylation of cTnI and C-protein was associated with the predicted increase in sensitivity to Ca 2ϩ . Our data provide evidence for a novel pathway of phosphatase regulation in cardiac myocytes.

P21 activated kinases

“…Interestingly, Pak1 is also associated with the sarcomere. In fact, Pak1 has been shown to localize to the sarcomeric Z disc, 81 though a precise role for Pak proteins in sarcomeric structure has not been defined.…”

A change of heart: oxidative stress in governing muscle function?

“…However, it may not have a direct effect on myofilament proteins, since myofilament protein phosphorylation was reported to be preserved in transgenic mouse models with altered calcineurin activity (Wilkins and Molkentin 2002). However, hypophosphorylation of several myofilament proteins has been attributed to PP1 and 2A (Ke et al 2004;Humphries et al 2002;Wu and Solaro 2007). Oxidative stress activates stress kinases, such as several PKC isoforms, PKD, CaMKII and P38 mitogen-activated protein kinases and inactivate PP1 and/or PP2A activity (Jin Jung et al 2013).…”

A preferred AMPK phosphorylation site adjacent to the inhibitory loop of cardiac and skeletal troponin I

“…54 Buscemi et al 25 found that phosphorylation of cTnI Ser149 by PAK3 was accompanied by an increase in the Ca 2þ sensitivity of myofilament force development. A study by Ke et al 55 found that, although PAK1 could phosphorylate cTnI in vitro, adenovirus infection of isolated myocytes with a constitutively active PAK1 construct decreased the overall phosphorylation status of cTnI and increased the Ca 2þ sensitivity of force production. 55 Because phosphorylation status of individual cTnI sites was not assessed in that study, it is not known if some sites underwent increased phosphorylation while others exhibited a proportionally greater decrease in dephosphorylation.…”

“…A study by Ke et al 55 found that, although PAK1 could phosphorylate cTnI in vitro, adenovirus infection of isolated myocytes with a constitutively active PAK1 construct decreased the overall phosphorylation status of cTnI and increased the Ca 2þ sensitivity of force production. 55 Because phosphorylation status of individual cTnI sites was not assessed in that study, it is not known if some sites underwent increased phosphorylation while others exhibited a proportionally greater decrease in dephosphorylation. Alternatively, different isoforms of PAK may have different effects on cTnI phosphorylation.…”