The PHLPP1 N-Terminal Extension Is a Mitotic Cdk1 Substrate and Controls an Interactome Switch

ABSTRACT

PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1) is a tumor suppressor that directly dephosphorylates a wide array of substrates, most notably the prosurvival kinase Akt. However, little is known about the molecular mechanisms governing PHLPP1 itself. Here, we report that PHLPP1 is dynamically regulated in a cell cycle-dependent manner and deletion of PHLPP1 results in mitotic delays and increased rates of chromosomal segregation errors. We show that PHLPP1 is hyperphosphorylated during mitosis by Cdk1 in a functionally uncharacterized region known as the PHLPP1 N-terminal extension (NTE). A proximity-dependent biotin identification (BioID) interaction screen revealed that during mitosis, PHLPP1 dissociates from plasma membrane scaffolds, such as Scribble, by a mechanism that depends on its NTE and gains proximity to kinetochore and mitotic spindle proteins such as KNL1 and TPX2. Our data are consistent with a model in which phosphorylation of PHLPP1 during mitosis regulates binding to its mitotic partners and allows accurate progression through mitosis. The finding that PHLPP1 binds mitotic proteins in a cell cycle- and phosphorylation-dependent manner may have relevance to its tumor-suppressive function.

KEYWORDS:

• PHLPP
• Cdk1
• mitosis
• phosphatase

SUPPLEMENTAL MATERIAL

Supplemental material is available online only.

ACKNOWLEDGMENTS

We thank members of the Newton and Dixon laboratories for helpful discussion. We kindly thank Hannah Tovell for assistance with revisions and Sourav Banerjee (UCSD) for performing FACS for live-cell imaging experiments. We thank Lloyd Trotman’s lab (CSHL) for the WT and Phlpp1^−/− MEFs, Kun Liang Guan’s lab (UCSD) for the HEK-293A cells, and Karen Oegema’s lab (UCSD) for the RPE1 cells and the pCDH-EF1 vector. We thank Dario Alessi’s lab (University of Dundee) for the PhosTag reagent, Robert Tukey’s lab (UCSD) for the SB203580 inhibitor, and Michael Karin’s lab (UCSD) for the JNK-IN-8 inhibitor and the pJNK antibody. We thank Dennis Young (Moore’s Cancer Center, UCSD) for help with analysis of flow cytometry data.

This work was supported by NIH R35 GM122523 (A.C.N.), NIH GM067946 (A.C.N.), NIH R01 GM074215 (A.D.) and the Canadian Institutes of Health Research (CIHR FDN 144301 to A.-C.G.). BioID experiments were performed at the Network Biology Collaborative Centre at the Lunenfeld-Tanenbaum Research Institute, a facility supported by Canada Foundation for Innovation funding, by the Ontarian Government and by Genome Canada and Ontario Genomics (OGI-139). Flow cytometry was performed at the Flow Cytometry Shared Resource Service at the Moore’s Cancer Center (UCSD). A.T.K. was supported in part by the University of California, San Diego Graduate Training Grant in Cellular and Molecular Pharmacology through National Institutes of Health Institutional Training Grant T32 GM007752 from the NIGMS. A.-C.G. is the Canada Research Chair (Tier 1) in Functional Proteomics.

Author contributions are as follows: A.T.K., C.W., G.L., and C.C.K. performed the experiments. C.W. designed and performed the BioID screen under the supervision of A.-C.G. C.C.K. performed the phospho-mass spectrometry. G.L. performed experiments whose results are shown in Fig. 2H and I, Fig. 3C, and Fig. 6. A.T.K. performed the remaining experiments and generated the figures. P.L.-G. and A.D. provided assistance and equipment for time-lapse fluorescence microscopy. A.T.K. and A.C.N. wrote the manuscript, and all authors edited the manuscript. A.T.K. and A.C.N. conceived the project.