NIPA Phosphorylation and Inactivation at G2/M Is Mediated by ERK2.

Abstract 2513

Poster Board II-490

Regulated oscillation of protein expression is an essential mechanism of cell cycle control. The SCF class of E3 ubiquitin ligases is involved in this process by targeting cell cycle regulatory proteins for degradation by the proteasome. We previously reported cloning of NIPA (Nuclear Interaction Partner of ALK) in complex with constitutively active oncogenic fusions of ALK, contributing to the development of lymphomas and sarcomas. Subsequently we characterized NIPA as a F-Box protein that defines an oscillating ubiquitin E3-ligase. The SCF-NIPA complex targets nuclear cyclin B1 for ubiquitination in interphase while phosphorylation of NIPA in late G2 phase and mitosis inactivates the complex to allow for accumulation of cyclin B1, a critical event for proper G2/M transition. Thus, SCF-NIPA executes an important G2/M checkpoint control. We recently specified three serine residues Ser 354, 359 and 395 implicated in NIPA phosphorylation at G2/M. These data suggest a sequential NIPA phosphorylation, where initial Ser 354 and 359 phosphorylation is most crucial for SCF-NIPA inactivation by dissociating the SCF-NIPA complex. Here we aimed to find the kinase responsible in this initial most important phosphorylation step. Using in vitro kinase assays we identified both ERK1 and ERK2 to phosphorylate NIPA with high efficiency. Mutation of either Ser 354 or Ser 359 abolished ERK-dependent NIPA phosphorylation. Inhibition of ERK1/2 activity in cell lines by specific inhibitors resulted in decreased NIPA phosphorylation at G2/M. To differentiate between phosphorylation by ERK1 and ERK2, we combined cell cycle analysis with stable expression of microRNA's targeting both isoforms. To this end NIH/3T3 cells were retrovirally transduced with microRNAs targeting ERK1 and 2 and cell cycle progression was analysed by BRDU/PI labeling. Using this approach, we are able to show that ERK2 but not ERK1 mediates NIPA phosphorylation at G2/M. Furthermore, ERK2 silencing leads to a distinct phenotype in cell cycle progression with a delay of ERK2 knockdown cells at the G2/M transition. Thus, our data indicate, that the recently described divergent functions of ERK1 and ERK2 in cell cycle regulation could be in part due to the differential ability of these kinases to phosphorylate and inactivate NIPA at G2/M. Since checkpoint proteins such as NIPA are constitutively inactivated in tumor cells ERK2 might represent an interesting target to reconstitute important cell cycle checkpoint controls in malignant cells.