Identification and characterization of a novel alpha-kinase with a von Willebrand factor A-like motif localized to the contractile vacuole and Golgi complex in Dictyostelium discoideum.
We have identified a new protein kinase in Dictyostelium discoideum that carries the same conserved class of "alpha-kinase" catalytic domain as reported previously in myosin heavy chain kinases (MHCKs) in this amoeba but that 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 than previously characterized alpha kinases in this organism, which all seem to be MHCKs.
Betapudi
Venkaiah
V
Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH 44106-4970, USA.
Mason
Cynthia
C
Licate
Lucila
L
Egelhoff
Thomas T
TT
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002147:Calmodulin / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D004023:Dictyostelium / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D015870:Gene Expression; D017125:Genes, Protozoan; D006056:Golgi Apparatus / Q000201:enzymology; D008969:Molecular Sequence Data; D024462:Myosin Type II / Q000378:metabolism; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014617:Vacuoles / Q000201:enzymology; D014841:von Willebrand Factor / Q000737:chemistry / Q000235:genetics
D002147:Calmodulin; D018076:DNA, Complementary; D016054:DNA, Protozoan; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D014841:von Willebrand Factor; D011494:Protein Kinases; D024462:Myosin Type II
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