Phosphorylation of yeast hexokinase 2 regulates its nucleocytoplasmic shuttling.
Nucleocytoplasmic shuttling of Hxk2 induced by glucose levels has been reported recently. Here we present evidence that indicates that Hxk2 nucleocytoplasmic traffic is regulated by phosphorylation and dephosphorylation at serine 14. Moreover, we identified the protein kinase Snf1 and the protein phosphatase Glc7-Reg1 as novel regulatory partners for the nucleocytoplasmic shuttling of Hxk2. Functional studies revealed that, in contrast to the wild-type protein, the dephosphorylation-mimicking mutant of Hxk2 retains its nuclear localization in low glucose conditions, and the phosphomimetic mutant of Hxk2 retains its cytoplasmic localization in high glucose conditions. Interaction experiments of Hxk2 with Kap60 and Xpo1 indicated that nuclear import of the S14D mutant of Hxk2 is severely decreased but that the export is significantly enhanced. Conversely, nuclear import of the S14A mutant of Hxk2 was significantly enhanced, although the export was severely decreased. The interaction of Hxk2 with Kap60 and Xpo1 was found to occur in the dephosphorylated and phosphorylated states of the protein, respectively. In addition, we found that Hxk2 is a substrate for Snf1. Mutational analysis indicated that serine 14 is a major in vitro and in vivo phosphorylation site for Snf1. We also provide evidence that dephosphorylation of Hxk2 at serine 14 is a protein phosphatase Glc7-Reg1-dependent process. Taken together, this study establishes a functional link between Hxk2, Reg1, and Snf1 signaling, which involves the regulation of Hxk2 nucleocytoplasmic shuttling by phosphorylation-dephosphorylation of serine 14.
Department of Biochemistry and Molecular Biology, University of Oviedo, 33006 Oviedo, Spain.
Paula
P
Fernández-García
Rafael
R
Peláez
Pilar
P
Herrero
Fernando
F
Moreno
D021581:Active Transport, Cell Nucleus / Q000502:physiology; D019943:Amino Acid Substitution; D002467:Cell Nucleus / Q000201:enzymology* / Q000235:genetics; D003593:Cytoplasm / Q000201:enzymology* / Q000235:genetics; D006593:Hexokinase / Q000235:genetics / Q000378:metabolism*; D028884:Karyopherins / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D010766:Phosphorylation / Q000502:physiology; D054645:Protein Phosphatase 1 / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D000097606:Exportin 1 Protein
D006593:Hexokinase; D028884:Karyopherins; D054645:Protein Phosphatase 1; D017346:Protein Serine-Threonine Kinases; D018160:Receptors, Cytoplasmic and Nuclear; D029701:Saccharomyces cerevisiae Proteins; D000097606:Exportin 1 Protein; C071570:SNF1-related protein kinases; C490214:HXK2 protein, S cerevisiae; C092048:GLC7 protein, S cerevisiae; C070829:REG1 protein, S cerevisiae