In this work an immunochemical, radioactive labelling and activity analysis of the PKA/AKAP complex submitochondrial localization in rat heart is presented. The densitometric immunoblot analysis shown that the large majority (90%) of mitochondrial PKA is found in the mitoplast fraction (inner membrane/matrix fraction) when prepared both from isolated mitochondria or cardiomyocytes. The remaining amount of C-PKA and R-PKA, associated with the outer mitochondrial membrane is, thus relatively small. The same distribution pattern applies to AKAP121. This distribution pattern was further verified by measurement of the specific PKA activity. C-PKA, R-PKA and AKAP121 are largely resistant to trypsin digestion, unless mitochondria are not disrupted by Triton-X 100. R-PKA and with it C-PKA are released from mitoplasts by the Ht31 peptide, competitive inhibitor for the R-PKA-AKAP binding. Electron microscopy analysis of proteins labelled by gold-conjugated antibody shows that like C-PKA and R-PKA (4), also AKAP121 is present in the inner mitochondrial compartment apparently clustered on the inner mitochondrial membrane. Radiolabelling analysis of R-PKA by 32P-cAMP and AKAP by 32P-phosphorylated RII-PKA respectively, confirmed the presence of these proteins in the inner mitochondrial compartment and revealed, at the same time, that in the inner mitochondrial compartment R-PKA and AKAP undergo proteolytic degradation by mitochondrial processing peptidase. Turnover of these proteins might contribute to mid-term plasticity of the cAMP-controlled protein phosphorylation system. A down-stream extension of the effect of cAMP in the inner mitochondrial compartment mediated by adenylyl cyclase, phosphodiesterases, PKA, protein phosphatase(s), EPAC proteins and G proteins localized in this subcellular compartment can have definite impact on cellular energy metabolism, apoptosis, cell growth, differentiation and transformation.
cAMP-dependent protein kinase and a-kinase anchor proteins in the inner compartment of mammalian heart mitochondria / A., Sardanelli; A., Signorile; R., Nuzzi; D., DE RASMO; Z., TECHNIKOVA DOBROVA; Z., Drahota; Pica, Alessandra; Occhiello, Antonella; S., Papa. - STAMPA. - Suppl 14:(2006), pp. 125-125. (Intervento presentato al convegno From molecules to physiology and pathology. Proceedings of the 14th European Bioenergetics Conference tenutosi a Mosca, Russia nel July 22-27, 2006).
cAMP-dependent protein kinase and a-kinase anchor proteins in the inner compartment of mammalian heart mitochondria
PICA, ALESSANDRA;OCCHIELLO, ANTONELLA;
2006
Abstract
In this work an immunochemical, radioactive labelling and activity analysis of the PKA/AKAP complex submitochondrial localization in rat heart is presented. The densitometric immunoblot analysis shown that the large majority (90%) of mitochondrial PKA is found in the mitoplast fraction (inner membrane/matrix fraction) when prepared both from isolated mitochondria or cardiomyocytes. The remaining amount of C-PKA and R-PKA, associated with the outer mitochondrial membrane is, thus relatively small. The same distribution pattern applies to AKAP121. This distribution pattern was further verified by measurement of the specific PKA activity. C-PKA, R-PKA and AKAP121 are largely resistant to trypsin digestion, unless mitochondria are not disrupted by Triton-X 100. R-PKA and with it C-PKA are released from mitoplasts by the Ht31 peptide, competitive inhibitor for the R-PKA-AKAP binding. Electron microscopy analysis of proteins labelled by gold-conjugated antibody shows that like C-PKA and R-PKA (4), also AKAP121 is present in the inner mitochondrial compartment apparently clustered on the inner mitochondrial membrane. Radiolabelling analysis of R-PKA by 32P-cAMP and AKAP by 32P-phosphorylated RII-PKA respectively, confirmed the presence of these proteins in the inner mitochondrial compartment and revealed, at the same time, that in the inner mitochondrial compartment R-PKA and AKAP undergo proteolytic degradation by mitochondrial processing peptidase. Turnover of these proteins might contribute to mid-term plasticity of the cAMP-controlled protein phosphorylation system. A down-stream extension of the effect of cAMP in the inner mitochondrial compartment mediated by adenylyl cyclase, phosphodiesterases, PKA, protein phosphatase(s), EPAC proteins and G proteins localized in this subcellular compartment can have definite impact on cellular energy metabolism, apoptosis, cell growth, differentiation and transformation.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
