CDK1‐dependent Phosphorylation of the Tumor Suppressor Phosphatase, PHLPP1, Regulates the Mitotic PHLPP1 Interactome

Abstract

PH domain Leucine Rich Repeat Protein Phosphatase 1 (PHLPP1) is a tumor suppressor originally discovered for its ability to directly dephosphorylate and inactivate the pro‐survival kinase Akt, a key transducer of growth factor signaling. A number of other PHLPP1 targets have been identified, but still little is known about the molecular mechanisms governing the function and regulation of PHLPP1 itself. Here we report that PHLPP1 is hyperphosphorylated during mitosis in a CDK1‐dependent manner, and that this hyperphosphorylation regulates its interaction with mitotic proteins. Specifically, we show that PHLPP1 undergoes an electrophoretic mobility shift in mitotic cells that is lost with lambda phosphatase treatment, and is prevented by CDK1 inhibition. This mobility shift can be recreated in vitro using recombinant CDK1‐Cyclin B. Mass spectrometry and biochemical analysis reveals that these phosphorylations modify the N‐terminus of PHLPP1, a functionally uncharacterized region. A proximity dependent biotin identification (BioID) interaction screen revealed that mitotic PHLPP1 interacts with components of the mitotic spindle apparatus and the kinetochore. Additionally, the data suggest that the N‐terminus is required for the dissociation of PHLPP1 from interphase scaffolds, such as Scribble, during mitosis. During mitotic exit, PHLPP1 protein levels decrease, suggesting that PHLPP1 is degraded during mitotic exit. This correlates with an increase in Akt Ser473 phosphorylation, a validated cellular target of PHLPP1. Our data are consistent with a model in which phosphorylation of PHLPP1 during mitosis regulates binding to its mitotic partners and allows proper passage through mitosis. Reversible protein phosphorylation, orchestrated by kinases and phosphatases, plays a role in controlling proper progression through mitosis, which is essential as errors in mitosis can result in aneuploidy, a hallmark of cancer. The finding that PHLPP1 binds mitotic proteins in a cell cycle and phosphorylation‐dependent manner may have relevance to its tumor suppressive function.