Abstract 21319: Phospholemman Migrates out of Cardiomyocyte Caveolae upon Protein Kinase C Activation
Phospholemman (PLM), the principal sarcolemmal substrate for protein kinases A and C (PKA and PKC) in the heart, is a regulator of the cardiac sodium pump. We investigated the subcellular localisation and phosphorylation / dephosphorylation of PLM in freshly isolated adult rat ventricular myocytes (ARVM) by subcellular fractionation and phosphospecific immunoblotting. PKA phosphorylates PLM at serine 68, and PKC phosphorylates PLM at serines 63 and 68, and threonine 69 (S63, S68, T69). In unstimulated ARVM, PLM is substantially (30%) phosphorylated at S63 and S68. We investigated the sensitivity of the PLM phosphatase to okadaic acid. S63 dephosphorylation was ∼10x more sensitive to okadaic acid than S68 and T69 dephosphorylation (log IC50 (nM) okadaic acid for dephosphorylation S63 2.0±0.3 versus 3.1±0.2 and 3.6±0.2 for S68 and T69 respectively). Density gradient centrifugation of ARVM lysates indicated PLM and the cardiac sodium pump reside in caveolae. In ARVM treated with the PKC activating agent PMA (300nM), the caveolar content of PLM was reduced by 50%, suggesting PLM migrates out of caveolae upon phosphorylation. Since caveolae concentrate signalling molecules, we hypothesised that PLM localisation to caveolae may be required for its efficient phosphorylation and/or dephosphorylation. ARVM were depleted of caveolae by treatment with the cholesterol-depleting agent methyl-beta-cyclodextrin (MβCD 2mM, 1 hour). MβCD caused a substantial increase in the basal phosphorylation of S63, S68 and T69. We observed no differences in phosphorylation of any residues of PLM between control and MβCD-treated cells after PKC activation with PMA (300nM) or PKA activation with forskolin (10µM). In conclusion, the sensitivity of the PLM S63 phosphatase to okadaic acid is PP2A-like, whilst the S68 and T69 phosphatase is PP1-like. PLM may therefore provide a functional link between both PP1 and PP2A and the cardiac sodium pump. In unstimulated cardiac myocytes, dephosphorylation of PLM requires intact caveolae, and PKC activation is linked to PLM translocation out of caveolae into the bulk sarcolemma. Taken together, these data suggest that the presence of PLM in caveolae is dynamically regulated, and is a means by which its phosphorylation status is controlled.
- © 2010 by American Heart Association, Inc.