Nup53p is a target of two mitotic kinases, Cdk1p and Hrr25p.
Nuclear pore complexes (NPCs) form channels across the nuclear envelope and provide the sole sites of molecular exchange between the cytoplasm and nucleoplasm. The NPC is a target of a number of post-translational modifications, including phosphorylation, yet the functions of these modifications are ill defined. Here, we have investigated the mitotic specific phosphorylation of a yeast nucleoporin Nup53p. Two kinases were identified that phosphorylate Nup53p: the mitotic kinase Cdk1p/Cdc2p/Cdc28p and the casein kinase Hrr25p. Hrr25p was identified by screening 119 yeast kinases for their ability to phosphorylate Nup53p in vitro. Conditional alleles of Hrr25p support the conclusion that Hrr25p phosphorylates Nup53p in vivo. We further demonstrated using solution binding and affinity purification assays, that Hrr25p directly binds Nup53p in an interaction that is destabilized by the phosphorylation of Nup53p. Consistent with this observation, we observed that Hrr25p moves between distinct locations in the cell during the cell cycle including the nucleus, the cortex of the emerging bud and the spindle pole bodies. Cdk1p also contributes to Nup53p phosphorylation as specific inhibition of Cdk1p or mutation of Cdk1p consensus sites partially blocked its phosphorylation. The ability of nup53 alleles containing Cdk1p site mutations to complement synthetic defects of nup53 Delta nup170 Delta strains is linked to a function for Nup53p in the spindle assembly checkpoint.
Lusk
C Patrick
CP
Department of Cell Biology, University of Alberta, Edmonton, AB T6G 2H7, Canada.
Waller
Daniel D
DD
Makhnevych
Taras
T
Dienemann
Axel
A
Whiteway
Malcolm
M
Thomas
David Y
DY
Wozniak
Richard W
RW
D000483:Alleles; D016203:CDC2 Protein Kinase / Q000235:genetics / Q000378:metabolism*; D047389:Casein Kinase I / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D009154:Mutation; D028861:Nuclear Pore Complex Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D010957:Plasmids; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism
C116843:NUP53 protein, S cerevisiae; D028861:Nuclear Pore Complex Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D005982:Glutathione Transferase; D047389:Casein Kinase I; C070775:HRR25 protein, S cerevisiae; D016203:CDC2 Protein Kinase