Direct interactions with both p27 and Cdk2 regulate Spy1-mediated proliferation in vivo and in vitro

ABSTRACT

Families of cyclin-like proteins have emerged that bind and activate cyclin dependent kinases (Cdk)s, directing the phosphorylation of noncanonical Cdk substrates. One of these proteins, Spy1, has demonstrated the unique ability to directly bind and activate both Cdk1 and Cdk2, as well as binding and promoting the degradation of at least one Cdk inhibitor, p27^Kip1. Spy1 accelerates somatic cell growth and proliferation and is implicated in a number of human cancers including the breast, brain and liver. Herein we isolate key residues mediating the direct interaction with p27. We use mutants of Spy1 to determine the physiological role of direct interactions with distinct binding partners Cdk2 and p27. We demonstrate that disrupting the direct interaction with either Spy1 binding partner decreased endogenous activity of Cdk2, as well as Spy1-mediated proliferation. However, only the direct interaction with p27 was essential for Spy1-mediated effects on p27 stability. In vivo neither mutation completely prevented tumorigenesis, although each mutation slowed the rate of Spy1-mediated tumorigenesis and decreased overall tumor volumes. This work supports the conclusion that direct interaction with both p27 and Cdk2 contribute to Spy1-mediated effects on cell growth. It is important to elucidate the dynamics of these interactions and to consider these data when assessing functional outcomes.

Keywords:

• cell cycle, cell growth, senescence, tumorigenesis, RINGO

This article is referred to by:

The Speedy A, Cdk2, p27 triangle

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Dr. C. Shermanko for providing the HCll cell lines, Dr. B. Welm for providing lentivirus vector (PEIZ), Dr. D.J. Donoghue for supplying vectors and the Spy1 antibody and J. Maimaiti and N. Paquette for technical assistance.

Funding

BF was supported by a fellowship funded by the Canadian Breast Cancer Foundation-Ontario Region.

This study was supported by operating funds from the Canadian Cancer Society (CCS)/Canadian Breast Cancer Research Alliance (CBCRA) #020513.

Author contributions

Experiments were conceived and designed by MAS, BF, DM, LAP. Experiments were performed by MAS, BF and DM. Data was analyzed by MAS, BF, DM and LAP. Reagents/materials/tools provided by LAP. Manuscript was prepared by MAS, BF and LAP.