The Role Of PP2A B56? Regulatory Subunit In Cardiac Calcium Channel Cav1.2 Signaling Complex

The L-type Ca2+channel Cav1.2 forms macromolecular signaling complexes that comprise the ? adrenergic receptor,trimeric Gs protein,adenylyl cyclase,cAMP-denpendent protein kinase(PKA)and protein phosphatases 2A for efficient signaling in heart.PP2A counteracts increase in Cav1.2 channel activity and phosphorylation by PKA.PP2A is a serine/threonine-selective holoen2yme composed of a catalytic,scaffolding,and regulatory subunit.There are 13 known PP2A regulatory B subunits that target the phosphates activity to various cellular compartment or organelles,confer substrate specificity or control enzyme activity.Hall at all.reportes immunoprecipitation of Cav1.2 leads to coprecipitation of various B'(all isoforms)and PR59.The role of these B subunits in the Cav1.2 signaling complex is unknown.Recent years,because B subunits are differently expressed in many tissues and cell types and differently distribution patterns within cell types,more and more attention is paid to B subunits.B56? exhibits the highest expression in the heart muscle.B56? is localized to both M-line and Z-line in cardiomyocytes and Cav1.2 is known to be mainly localized in adult cardiomyocytes at Z-line.These imply that B56? may play a role in dephosphorylation of Cav1.2 by PP2A.Little et al.show that mice deficient in the PP2A regulatory B56? display increased PP2A activity suggesting the B56a regulatory subunit has an autoinhibitory role.Increased PP2A activity in B56?+/-myocytes results in reduced Ca2+waves and sparks.These also imply B56? may play an important role in dephosphorylation Cav1.2 by PP2A.So what is the role of B56? in dephosphorylation of Cav1.2 by PP2A?It is explored from two sides.The way of B56? interaction with Cav1.21 Co-imunoprecipitation was performed in adult mouse ventricular muscle lysis and H9c2 cell lysis,and the results confirmed that B56? was associated with PP2A/C and Cav1.2.2 Co-location of Cav1.2 and B56? was observed in either adult mouse ventricular myocytes or neonatal mouse ventricular myocytes.3 NT(1-154 aa),and Loopl(436-554 aa)and CT(1505-2171 aa)of Cav1.2 were cloned into pcDNA3.0-HA by molecular cloning technique.Co-imunoprecipitation assays in HEK293 cells co-expressing one of the fragments and B56? showed none of them interacted with B56?.GST pull dow assay showed that the interaction between B56? and Cav1.2 mediated by PP2A/C?.It suggested there was no direct interaction between Cav1.2 and B56?.The role of B56? in cardiac Cav1.2 signaling complex1 The H9c2 cells overexpression of B56? displayed 8 fold increase.The abundance of other subunits of PP2A was not altered.The overexprssion of B56? was associated with reduced PP2A activity suggesting the B56? regulatory subunit has an autoinhibitory role that suppressed excess PP2A activity.2 In the basal condition,the decrese in PP2A activity in the H9c2 cells with B56?overexpression resulted in increased phosphorylation of Cav1.2.3 The B56? overexpression augmented the effect of the stimulation of ?-adrenergic receptors by ISO on Cav1.2.4 Consistently,the overexprssion of B56? in HEK293 was associated with reduced PP2A activity suggesting the B56a regulatory subunit had an autoinhibitory role that suppressed excess PP2A activity.Taken together,our data demonstrated that there was no direct interaction between Cav1.2 and B56?,and the interaction between them mediated by PP2A/Ca in the heart.The overexprssion of B56? was associated with reduced PP2A activity and increased phosphorylation of Cav1.2 suggesting that an integral component of the PP2A homoenzyme had an important inhibitory role in controlling PP2A activity.Regulation of PP2A activity by B56? depended on the interaction between PP2A/C?and B56?.