LATS1 and LATS2 phosphorylate CDC26 to modulate assembly of the tetratricopeptide repeat subcomplex of APC/C.
In budding yeast, the Mitotic Exit Network (MEN) regulates anaphase promoting complex/cyclosome (APC/C) via the Dbf2-Cdc14 signaling cascade. Dbf2 kinase phosphorylates and activates Cdc14 phosphatase, which removes the inhibitory phosphorylation of the APC/C cofactor Cdh1. Although each component of the MEN was highly conserved during evolution, there is presently no evidence supporting direct phosphorylation of CDC14 by large tumor suppressor kinase 1 (LATS1), the human counterpart of Dbf2; hence, it is unclear how LATS1 regulates APC/C. Here, we demonstrate that LATS1 phosphorylates the Thr7 (T7) residue of the APC/C component CDC26 directly. Nocodazole-induced phosphorylation of T7 was reduced by knockdown of LATS1 and LATS2 in HeLa cells, indicating that both of these kinases contribute to the phosphorylation of CDC26 in vivo. The T7 residue of CDC26 is critical for its interaction with APC6, a tetratricopeptide repeat-containing subunit of APC/C, and mutation of this residue to Asp (T7D) reduced the interaction of CDC26 with APC6. Replacement of endogenous CDC26 in HeLa cells with exogenous phosphor-mimic T7D-mutated CDC26 increased the elution size of APC/C subunits in a gel filtration assay, implying a change in the APC/C assembly upon phosphorylation of CDC26. Furthermore, T7D-mutated CDC26 promoted the ubiquitination of polo-like kinase 1, a well-known substrate of APC/C. Overall, these results suggest that LATS1/2 are novel kinases involved in APC/C phosphorylation and indicate a direct regulatory link between LATS1/2 and APC/C.
Division of Gene Regulation, Institute for Advanced Medical Research, Keio University School of Medicine, Tokyo, Japan; Department of Obstetrics and Gynecology, Keio University School of Medicine, Tokyo, Japan.
Kenta
K
Masuda
Division of Gene Regulation, Institute for Advanced Medical Research, Keio University School of Medicine, Tokyo, Japan; Department of Obstetrics and Gynecology, Keio University School of Medicine, Tokyo, Japan.
Tatsuyuki
T
Chiyoda
Department of Molecular & Cellular BioAnalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan.
Naoyuki
N
Sugiyama
Division of Experimental Hematology & Cancer Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States of America.
Aldo
A
Segura-Cabrera
Department of Biochemistry, Keio University School of Medicine, Tokyo, Japan.
Yasuaki
Y
Kabe
Division of Gene Regulation, Institute for Advanced Medical Research, Keio University School of Medicine, Tokyo, Japan; Department of Obstetrics and Gynecology, Keio University School of Medicine, Tokyo, Japan.
Arisa
A
Ueki
Kouji
K
Banno
Department of Obstetrics and Gynecology, Keio University School of Medicine, Tokyo, Japan.
Koji
K
Banno
Department of Biochemistry, Keio University School of Medicine, Tokyo, Japan; CREST, Japan Science and Technology Agency, Tokyo, Japan.
Makoto
M
Suematsu
Department of Obstetrics and Gynecology, Keio University School of Medicine, Tokyo, Japan.
Daisuke
D
Aoki
Department of Molecular & Cellular BioAnalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan.
Yasushi
Y
Ishihama
Division of Gene Regulation, Institute for Advanced Medical Research, Keio University School of Medicine, Tokyo, Japan.
Hideyuki
H
Saya
Division of Gene Regulation, Institute for Advanced Medical Research, Keio University School of Medicine, Tokyo, Japan.
Shinji
S
Kuninaka
D000595:Amino Acid Sequence; D064173:Anaphase-Promoting Complex-Cyclosome / Q000737:chemistry / Q000378:metabolism*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D055503:Protein Multimerization; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D025521:Tumor Suppressor Proteins / Q000378:metabolism*
D025521:Tumor Suppressor Proteins; D064173:Anaphase-Promoting Complex-Cyclosome; C092745:LATS1 protein, human; C406449:LATS2 protein, human; D017346:Protein Serine-Threonine Kinases