Cell cycle-dependent phosphorylation of human CDC5 regulates RNA processing

Abstract

CDC5 proteins are components of the pre-mRNA splicing complex and essential for cell cycle progression in yeast, plants and mammals. Human CDC5 is phosphorylated in a mitogen-dependent manner, and its association with the spliceosome is ATP-dependent. Examination of the amino acid sequence suggests that CDC5L may be phosphorylated at up to 28 potential consensus recognition sequences for known kinases, however, the identity of actual phosphorylation sites, their role in regulating CDC5L activity, and the kinases responsible for their phosphorylation have not previously been determined. Using two-dimensional phosphopeptide mapping and nanoelectrospray mass spectrometry, we now show that CDC5L is phosphorylated on at least nine sites in vivo. We demonstrate that while CDC5L is capable of forming homodimers in vitro and in vivo, neither homodimerization nor nuclear localization is dependent on phosphorylation at these sites. Using an in vitro splicing assay, we show that phosphorylation of CDC5L at threonines 411 and 438 within recognition sequences for CDKs are required for CDC5L-mediated pre-mRNA splicing. We also demonstrate that a specific inhibitor of CDK2, CVT-313, inhibits CDC5L phosphorylation in both in vitro kinase assays and in vivo radiolabeling experiments in cycling cells. These studies represent the first demonstration of a regulatory role for phosphorylation of CDC5L, and suggest that targeting these sites or the implicated kinases may provide novel strategies for treating disorders of unguarded cellular proliferation, such as cancer.

Characterization of purified human Bact spliceosomal complexes reveals compositional and morphological changes during spliceosome activation and first step catalysis

“…3). Previous studies demonstrated that human CDC5L is phosphorylated at at least nine sites in vivo, and suggested that the phosphorylation of threonines at positions 411 and 438 are required for the first step of splicing in vitro (Graub et al 2008). Based on our data it is thus tempting to hypothesize that the phosphorylation of CDC5L plays an important role in the first step of splicing, whereas phosphorylation of SF3b155 may play an important role in spliceosome activation.…”

“…3B). As MS analyses did not provide any evidence for potential glycosylation, ubiquitinylation or SUMOylation of CDC5L, and CDC5L was previously reported to be phosphorylated in vivo (Bernstein and Coughlin 1997;Graub et al 2008), we tested whether this slowly migrating form of CDC5L is phosphorylated. For this purpose, we treated C complexes with calf intestine pyrophosphatase (CIP) prior to immunoblotting, and then probed with anti-CDC5L antibodies.…”

Differential DNA methylation and PM2.5 species in a 450K epigenome-wide association study

“…49,50 Cell division cycle 5-like (CDC5L) is a crucial regulator of cell cycle G2/ M progression and a component of pre-mRNA processing; hence, previous research has suggested it as a target for cancer therapy 50,51 RPS6KB2 is also highly active in the G2 and M phases. 49 The DAVID bioinformatics analysis also indicated that Fe and Ni were associated with type II diabetes mellitus, insulin signaling pathways, and the MAPK pathway.…”

Semiquantitative Proteomic Analysis of the Human Spliceosome via a Novel Two-Dimensional Gel Electrophoresis Method

“…Reversible protein phosphorylation plays a key role during spliceosome assembly and the catalytic steps of splicing (reviewed in refs 28, 37). Approximately one-third of the spliceosomal proteins identified in the present study were found to be moderately to heavily phosphorylated, including all whose phosphorylation or dephosphorylation is known to be functionally relevant during splicing, such as SR proteins (reviewed in references 28 and 37), U1-70K (39), and CDC5L (16). Although the intensity of phosphostaining of some proteins varied, in the vast majority of cases these changes simply reflected changes in the abundance of that protein from one spliceosomal complex to the next.…”