Regulation of sea urchin egg myosin by 53K, a myosin-binding protein

Date of Award




Degree Name

Doctor of Philosophy (Ph.D.)


Cell Biology and Anatomy

First Committee Member

David R. Burgess - Committee Chair


A myosin-binding protein is identified that mediates the low ionic strength solubility of myosin in extracts of unfertilized sea urchin eggs. The protein, termed 53K, possesses a subunit molecular weight on SDS-PAGE of 53 kD and an S value of 7. The association between myosin and 53K is demonstrated by their co-precipitation from extracts upon the addition of nucleotides and co-sedimention at 24S by sedimentation velocity centrifugation. Further, myosin is immunoprecipitated from extract with antibody to 53K, and 53K binds to a myosin affinity column. When extract is depleted of 53K, myosin precipitates out of extract in a nucleotide-independent manner. Reconstituting 53K and myosin partially restores the low ionic strength solubility demonstrated by myosin in fresh egg extracts.The binding site of 53K on myosin was examined to determine how the interaction between these two proteins might influence myosin's solubility. Egg myosin digested with papain yields discrete fragments representing head and rod portions. Blot overlay techniques established that 53K binds to the head-rod junction on the myosin molecule. Additionally, both cross-linking studies using myosin fragments and 53K, and immunogold labelling of 53K bound to myosin indicated binding of 53K to the head-rod junction of myosin. Therefore, 53K may exert an effect on myosin solubility dependent, in part, on is binding site on myosin.It was demonstrated that 53K possesses myosin light chain kinase activity. 53K phosphorylates the regulatory light chains of egg and chicken gizzard myosin and increases the actin-activated ATPase activity of both myosins by approximately three fold. Autophosphorylation of 53K occurs, and may indicate that phosphorylation regulates 53K activity. 53K kinase activity is independent of Ca$\sp{2+}$/CaM although a second kinase activity exists in extracts that is Ca$\sp{2+}$/CaM-dependent. Contraction of reconstituted egg actin gels is enhanced by myosin light chain phosphorylation which is likely mediated by 53K. The myosin-binding protein 53K appears to be bifunctional in nature. Apart from the ability of 53K to mediate the low ionic strength solubility of myosin, it is also capable of myosin light chain phosphorylation. These two functional aspects of 53K may be closely linked in regulating myosin in the egg cytoplasm.


Biology, Anatomy

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