Phosphorylation of human cardiac troponin I G203S and K206Q linked to familial hypertrophic cardiomyopathy affects actomyosin interaction in different ways.
cAMP-dependent protein kinase (PKA)-dependent phosphorylation of the two serine residues in the amino terminal region unique to cardiac troponin I (cTnI) is known to cause two effects: (i) decrease of the maximum Ca2+-controlled thin filament-activated myosin S1-ATPase (actoS1-ATPase) activity and mean sliding velocity of reconstituted thin filaments; (ii) rightward shift of the Ca2+ activation curves of actoS1-ATPase activity, filament sliding velocity, and force generation. We have studied the influence of phosphorylation of human wild-type cTnI and of two mutant cTnI (G203S and K206Q) causing familial hypertrophic cardiomyopathy (fHCM) on the secondary structure by circular dichroism spectroscopy and on the Ca2+ regulation of actin-myosin interaction using actoS1-ATPase activity and in vitro motility assays. Both mutations slightly influence the backbone structure of cTnI but only the secondary structure of cTnI-G203S is also affected by bis-phosphorylation of cTnI. In functional studies, cTnI-G203S behaves similarly to wild-type cTnI, i.e. the mutation itself has no measurable effect and bis-phosphorylation alters the actoS1-ATPase activity and the in vitro thin filament motility in the same way as does bis-phosphorylation of wild-type cTnI. In contrast, the mutation K206Q leads to a considerable increase in the maximum actoS1-ATPase activity as well as filament motility compared to wild-type cTnI. Bis-phosphorylation of this mutant cTnI still suppresses the maximum actoS1-ATPase activity and filament sliding velocity but does no longer affect the Ca2+ sensitivity of these processes. Thus, these two fHCM-linked cTnI mutations, although reflecting similar pathological situations, exert different effects on the actomyosin system per se and in response to bis-phosphorylation of cTnI.
St. Josef-Hospital, Klinik der Ruhr-Universität Bochum, Gudrunstrasse 56, 44791 Bochum, Germany.
Yi
Y
Deng
Anja
A
Schmidtmann
Sebastian
S
Kruse
Vladimir
V
Filatov
Ludwig M G
LM
Heilmeyer
Kornelia
K
Jaquet
Rolf
R
Thieleczek
D008841:Actin Cytoskeleton / Q000378:metabolism; D000205:Actomyosin / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D024741:Cardiomyopathy, Hypertrophic, Familial / Q000235:genetics* / Q000378:metabolism*; D002942:Circular Dichroism; D006801:Humans; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D009206:Myocardium / Q000201:enzymology / Q000378:metabolism; D010766:Phosphorylation; D017433:Protein Structure, Secondary; D011817:Rabbits; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D019210:Troponin I / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D019210:Troponin I; D012694:Serine; D000205:Actomyosin; D000251:Adenosine Triphosphatases; D002118:Calcium