Specific degradation of troponin T and I by mu-calpain and its modulation by substrate phosphorylation.
The degradation of troponin (Tn) subunits by calpain was studied by incubating either isolated cardiac Tns or myocardial cryosections with two different calpain isoenzymes isolated from rat skeletal muscle. Western-blot analysis with monoclonal antibodies against TnI and TnT showed that mu-calpain was at least ten times more active than m-calpain in degrading TnI and TnT both in vitro and in situ. TnC was completely resistant to both proteinase forms. Phosphorylation by cyclic AMP-dependent protein kinase (PKA) isolated from rat skeletal muscle reduced the sensitivity of TnI to degradation. This effect in combination with an increased efficiency of the endogenous inhibitor [Salamino, De Tullio, Michetti, Mengotti, Melloni and Pontremoli (1994) Biochem. Biophys. Res. Commun. 199, 1326-1332] probably reduces the proteolytic activity of calpain in cells on PKA stimulation. Conversely, phosphorylation by protein kinase C (PKC) resulted in a twofold increase in the degradation of TnI. Degradation by m-calpain was not modified by Tn phosphorylation. The different sensitivity to mu-calpain might be related to changes in TnI oligomeric structure. Indeed, on PKC phosphorylation, the apparent molecular mass of TnI calculated from the distribution coefficient of Tn complex in Sephadex G-100 matrix was reduced from 90 to 30 kDa suggesting dissociation of the Tn complex.
Di Lisa
F
F
Dipartimento di Chimica Biologica, Università di Padova, Italy.
De Tullio
R
R
Salamino
F
F
Barbato
R
R
Melloni
E
E
Siliprandi
N
N
Schiaffino
S
S
Pontremoli
S
S
D000818:Animals; D002154:Calpain / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000378:metabolism; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology; D009206:Myocardium / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D013329:Structure-Activity Relationship; D014336:Troponin / Q000378:metabolism*; D019210:Troponin I; D020107:Troponin T
D007527:Isoenzymes; D010750:Phosphoproteins; D014336:Troponin; D019210:Troponin I; D020107:Troponin T; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D002154:Calpain