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MBC in Press, published online ahead of print February 23, 2005
Mol. Biol. Cell 10.1091/mbc.E04-07-0639

A more recent version of this article appeared on May 1, 2005
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Submitted on July 28, 2004
Revised on February 3, 2005
Accepted on February 15, 2005

Identification and Characterization of a Novel Alpha Kinase with a vWFA-like Motif Localized to the Contractile Vacuole and Golgi Complex in Dictyostelium discoideum

Venkaiah Betapudi, Cynthia Mason, Lucila Licate, and Thomas T. Egelhoff

Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH 44106-4970

Monitoring Editor: Peter Devreotes

We have identified a new protein kinase in Dictyostelium discoideum which carries the same conserved class of "alpha kinase" catalytic domain as previously reported in myosin heavy chain kinases (MHCKs) in this amoeba, but which has a completely novel domain organization. The protein contains an N-terminal von Willebrand Factor A (vWFA)-like motif, and is therefore named VwkA. Manipulation of VwkA expression level via high copy number plasmids (VwkA++ cells) or gene disruption (vwkA null cells) results in an array of cellular defects, including impaired growth and multinucleation in suspension culture, impaired development, and alterations in myosin II abundance and assembly. Despite sequence similarity to MHCKs, the purified protein failed to phosphorylate myosin II in vitro. Autophosphorylation activity however, was enhanced by calcium/calmodulin, and the enzyme can be precipitated from cellular lysates with calmodulin-agarose, suggesting that VwkA may directly bind calmodulin. VwkA is cytosolic in distribution, but enriched on the membranes of the contractile vacuole and Golgi-like structures in the cell. We propose that VwkA likely acts indirectly to influence myosin II abundance and assembly behavior, and possibly has broader roles the previously characterized alpha kinases in this organism which all appear to be MHC kinases.

Address correspondence to: Thomas T. Egelhoff (tte{at}case.edu)






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