Molecular mechanisms orchestrating cyclin stability.

A fundamental abnormality in cancer is the unchecked proliferation of cells. Rather than responding appropriately to signals that control normal cell division, cancer cells grow and divide in an uncontrolled manner, invade normal tissues and organs, and eventually spread throughout the body. We have shown that PARK2 is a critical regulator of the core cell cycle machinery that controls cell proliferation. PARK2, a gene on chromosome 6q25.2-q27 originally discovered as a frequent cause of autosomal recessive juvenile parkinsonism (ARJP), has previously been identified as a tumor suppressor gene by our lab.1 Recently, through genetic analysis of approximately 5,000 tumors samples across 11 tumor types, we found that PARK2 undergoes copy number loss in about 30% of tumors, with deletions occurring most often in serous ovarian, breast, colon, and bladder carcinomas. Intriguingly, PARK2 genetic loss was mutually exclusive with amplification of the genes encoding cyclin D1 (CCND1), cyclin E1 (CCNE1), and CDK4, suggesting that PARK2 and these cell cycle machinery components interact in a common pathway.2 Aside from these results, our study dramatically demonstrates the power of in silico pan-cancer genetic analysis for elucidating novel biology. PARK2 functions as an E3 ligase for ubiquitin-mediated proteolysis.3 Our experiments demonstrated that PARK2 depletion leads to abnormal accumulation of all cyclin D and E isoforms.2 Furthermore, cancer-related mutations of PARK2 abrogated its activity to regulate cyclin D and E stability. PARK2 degradation of cyclin D occurred through proteasome-mediated ubiquitination, and this process was dependent on phosphorylation of cyclin D1 at threonine 286. Strikingly, using pulse-chase experiments, we showed that PARK2 was necessary and sufficient to dictate cyclin D half-life even in the presence of other Fbox proteins that have been proposed to target cyclin D. The elucidation of the core cell cycle machinery and how this machinery is regulated has spanned more than 3 decades. One of the most challenging efforts has been to understand the mechanisms governing cyclin stability and coordination of stability across related cyclin classes. Proper targeting of cyclins for degradation is required for normal cell cycle control. This is performed by a number of ubiquitin E3 ligases. The Skp/Cullin/Fbox complexes (SCF) regulate progression through G1 and entry into S phase by degrading G1 cyclin-dependent kinase inhibitors (CKIs) and G1 cyclins.4 A few candidate E3 ligases have been suggested to target G1 cyclins for proteolysis. For example, FBX4 (which is a component of the SCFFBX4/αB-crystallin complex) targets and ubiquitinates cyclin D, whereas cyclin E ubiquitination is directed by the SCFFBW7 complex.5,6 Prior to our work, the mechanisms underlying the orchestration of different types of cyclins were unknown. Our work showed that PARK2 targets both cyclins D and E to regulate their stability. We discovered that PARK2 is a component of a new class of novel SCF-like ubiquitin complexes, PCF4 and PCF7, which regulate the degradation of cyclins D and E respectively. PARK2 binds to its substrate cyclin D and other PCF4 members (Cullin-1, αB-crystallin, and FBX4). Similarly, PARK binds to FBXW7 and Cullin, forming the PCF7 complex, which targets cyclin E for destruction. Interestingly, we found that PARK2 and FBX4 act in a synergistic manner to target cyclin D1. When both PARK2 and FBX4 are inactivated, cyclin D accumulation is dramatically greater than would be predicted by a simple additive model. And, combining recombinant PARK2 with FBX4 in vitro synergistically enhances overall E3 ligase activity. Accordingly, a recent report suggested that SCFFBX4 alone is not sufficient to control cyclin D1 stability.7 Our study shows that PARK2 is a master orchestrator of G1/S cyclin stability. PARK2 functions as a component of E3 ubiquitin ligase complexes that drive cyclin D and E degradation. This model is supported by a convergence of data from pan-cancer genetic analysis, functional genomic analysis, and biochemical dissection (Fig. 1). It would seem that PARK2 functions in a manner analogous to that of the cyclin dependent kinase inhibitor CDKN2A (p16). Whereas p16 controls the activity of the G1/S cyclins by binding and inhibiting the enzymatic activities of cyclin/CDK complexes, PARK2 inhibits G1/S progression by acting at the level of cyclin stability. These findings answer long-sought questions about how coordination of cyclin levels occurs but open up many new questions. How is PARK2 enzymatic activity post-translationally regulated? What is the biochemical mechanism of synergy between PARK2 and FBX4? How does CDKN2A and PARK2 work together to regulate G1/S progression? And, how do we exploit PARK2 deficiency in cancer therapy? Future studies will be needed to answer these important questions. Figure 1. PARK2 inactivation and cell cycle dysfunction. PARK2 helps control levels of cyclin D and E by targeting them for degradation. In cancer cells that have lost PARK2, cyclin D and E accumulate, leading to increased phosphorylation of Rb and cell cycle progression. ...