Phosphorylation by casein kinase 2 regulates Nap1 localization and function.
In Saccharomyces cerevisiae, the evolutionarily conserved nucleocytoplasmic shuttling protein Nap1 is a cofactor for the import of histones H2A and H2B, a chromatin assembly factor and a mitotic factor involved in regulation of bud formation. To understand the mechanism by which Nap1 function is regulated, Nap1-interacting factors were isolated and identified by mass spectrometry. We identified several kinases among these proteins, including casein kinase 2 (CK2), and a new bud neck-associated protein, Nba1. Consistent with our identification of the Nap1-interacting kinases, we showed that Nap1 is phosphorylated in vivo at 11 sites and that Nap1 is phosphorylated by CK2 at three substrate serines. Phosphorylation of these serines was not necessary for normal bud formation, but mutation of these serines to either alanine or aspartic acid resulted in cell cycle changes, including a prolonged S phase, suggesting that reversible phosphorylation by CK2 is important for cell cycle regulation. Nap1 can shuttle between the nucleus and cytoplasm, and we also showed that CK2 phosphorylation promotes the import of Nap1 into the nucleus. In conclusion, our data show that Nap1 phosphorylation by CK2 appears to regulate Nap1 localization and is required for normal progression through S phase.
Calvert
Meredith E K
ME
Center for Cell Signaling, University of Virginia, Box 800577 HSC, Charlottesville, VA 22908, USA.
Keck
Kristin M
KM
Ptak
Celeste
C
Shabanowitz
Jeffrey
J
Hunt
Donald F
DF
Pemberton
Lucy F
LF
D021581:Active Transport, Cell Nucleus; D047390:Casein Kinase II / Q000378:metabolism*; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D019926:Cyclin B / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism*; D056490:Nucleosome Assembly Protein 1; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism
C093749:CLB2 protein, S cerevisiae; D018797:Cell Cycle Proteins; D019926:Cyclin B; D006657:Histones; C533556:NAP1 protein, S cerevisiae; D009687:Nuclear Proteins; D056490:Nucleosome Assembly Protein 1; D010750:Phosphoproteins; D029701:Saccharomyces cerevisiae Proteins; D047390:Casein Kinase II