Regulated phosphorylation of budding yeast's essential myosin V heavy chain, Myo2p.
The tail of the yeast myosin V encoded by Myo2p is known to bind several receptors for cargo delivery along polarized actin cables. However, it is not known how Myo2p activity is regulated or how it selects between cargoes. Here we show that Myo2p is reversibly phosphorylated in vivo. A short peptide at the N-terminal end of the cargo-binding domain contains three residues contributing to single or doubly phosphorylated species. We confirm that the tail consists of two proteolytically resistant subdomains and identify a functionally important region N-terminal to subdomain 1 that includes the phosphorylation sites. Mutagenesis of the phosphorylation sites to alanine abolished a mobility shift diagnostic of phosphorylation, whereas mutagenesis to glutamic acid produced the shift and the formation of an additional phosphorylated species. These substitutions did not affect overall cell growth. However, one of the sites is predicted to be a substrate of cAMP-dependent protein kinase (PKA), and yeast expressing Myo2p with alanine substitutions is resistant to otherwise lethal overexpression of PKA, whereas the glutamic acid mutant is supersensitive to overexpression of PKA. These results suggest that in yeast, Myo2p is subject to phosphoregulation involving a PKA-related signaling pathway.
Department of Molecular Biology and Genetics, Biotechnology Building, Cornell University, Ithaca, NY 14853, USA.
Aster
A
Legesse-Miller
Sheng
S
Zhang
Felipe H
FH
Santiago-Tirado
Colleen K
CK
Van Pelt
Anthony
A
Bretscher
D000409:Alanine / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence / Q000235:genetics; D019943:Amino Acid Substitution; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D009154:Mutation; D018995:Myosin Heavy Chains / Q000235:genetics / Q000378:metabolism*; D024701:Myosin Type V / Q000235:genetics / Q000378:metabolism*; D010641:Phenotype; D010766:Phosphorylation; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation
C077512:MYO2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; D024701:Myosin Type V; D018995:Myosin Heavy Chains; D000409:Alanine