The kinase domain alters the kinetic properties of the myosin IIIA motor.
Myosin IIIA is unique among myosin proteins in that it contains an N-terminal kinase domain capable of autophosphorylating sites on the motor domain. A construct of myosin IIIA lacking the kinase domain localizes more efficiently to the stereocilia tips and alters the morphology of the tips in inner ear hair cells. Therefore, we performed a kinetic analysis of myosin IIIA without the kinase domain (MIII DeltaK) and compared these results with our reported analysis of myosin IIIA containing the kinase domain (MIII). The steady-state kinetic properties of MIII DeltaK indicate that it has a 2-fold higher maximum actin-activated ATPase rate (kcat = 1.5 +/- 0.1 s-1) and a 5-fold tighter actin affinity (KATPase = 6.0 +/- 1.4 microM, and KActin = 1.4 +/- 0.4 microM) compared to MIII. The rate of ATP binding to the motor domain is enhanced in MIII DeltaK (K1k+2 approximately 0.10 +/- 0.01 microM-1.s-1) to a level similar to the rate of binding to MIII in the presence of actin. The rate of ATP hydrolysis in the absence of actin is slow and may be rate limiting. Actin-activated phosphate release is identical with and without the kinase domain. The transition between actomyosin.ADP states, which is rate limiting in MIII, is enhanced in MIII DeltaK. MIII DeltaK accumulates more efficiently at the tips of filopodia in HeLa cells. Our results suggest a model in which the activity and concentration of myosin IIIA localized to the tips of actin bundles mediates the morphology of the tips in sensory cells.
Dosé
Andréa C
AC
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California 94720, USA.
Ananthanarayanan
Shobana
S
Moore
Judy E
JE
Corsa
Amoreena C
AC
Burnside
Beth
B
Yengo
Christopher M
CM
D000199:Actins / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008954:Models, Biological; D009068:Movement / Q000502:physiology; D018995:Myosin Heavy Chains / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D025102:Myosin Type III / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D009475:Neurons, Afferent / Q000378:metabolism; D010770:Phosphotransferases / Q000378:metabolism / Q000502:physiology; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D011817:Rabbits; D018411:Spodoptera; D014162:Transfection
D000199:Actins; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; C491724:MYO3A protein, human; D000251:Adenosine Triphosphatases; D025102:Myosin Type III; D018995:Myosin Heavy Chains