High affinity Ca2+ binding sites of calmodulin are critical for the regulation of myosin Ibeta motor function.
We coexpressed myosin Ibeta heavy chain with three different calmodulin mutants in which the two Ca2+-binding sites of the two N-terminal domain (E12Q), C-terminal domain (E34Q), or all four sites (E1234Q) are mutated in order to define the importance of these Ca2+ binding sites to the regulation of myosin Ibeta. The calmodulin mutated at the two Ca2+ binding sites in N-terminal domain and C-terminal domain lost its lower affinity Ca2+ binding site and higher affinity Ca2+ binding site, respectively. We found that, based upon the change in the actin-activated ATPase activities and actin translocating activities, myosin Ibeta with E12Q calmodulin has the regulatory characteristics similar to myosin Ibeta containing wild-type calmodulin, while myosin Ibeta with E34Q or E1234Q calmodulin lose all Ca2+ regulation. While the increase in myosin Ibeta ATPase activity paralleled the dissociation of 1 mol of calmodulin from myosin Ibeta heavy chain for both wild type (above pCa 5) and E12Q calmodulin (above pCa 6), the Ca2+ level required for the inhibition of actin-translocating activity of myosin Ibeta was lower than that required for dissociation of calmodulin, suggesting that the conformational change induced by the binding of Ca2+ at the high affinity site but not the dissociation of calmodulin is critical for the inhibition of the motor activity. Our results suggest that the regulation of unconventional myosins by Ca2+ is directly mediated by the Ca2+ binding to calmodulin, and that the C-terminal pair of Ca2+-binding sites are critical for this regulation.
Zhu
T
T
Department of Physiology, University of Massachusetts Medical Center, Worcester, Massachusetts 01655, USA.
Beckingham
K
K
Ikebe
M
M
D000199:Actins / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D002147:Calmodulin / Q000235:genetics / Q000502:physiology*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002417:Cattle; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D018995:Myosin Heavy Chains / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism
D000199:Actins; D002135:Calcium-Binding Proteins; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D011994:Recombinant Proteins; D000251:Adenosine Triphosphatases; D018995:Myosin Heavy Chains; D002118:Calcium