The P Body Protein Dcp1a Is Hyper-phosphorylated during Mitosis

Abstract

Processing bodies (PBs) are non-membranous cytoplasmic structures found in all eukaryotes. Many of their components such as the Dcp1 and Dcp2 proteins are highly conserved. Using live-cell imaging we found that PB structures disassembled as cells prepared for cell division, and then began to reassemble during the late stages of cytokinesis. During the cell cycle and as cells passed through S phase, PB numbers increased. However, there was no memory of PB numbers between mother and daughter cells. Examination of hDcp1a and hDcp1b proteins by electrophoresis in mitotic cell extracts showed a pronounced slower migrating band, which was caused by hyper-phosphorylation of the protein. We found that hDcp1a is a phospho-protein during interphase that becomes hyper-phosphorylated in mitotic cells. Using truncations of hDcp1a we localized the region important for hyper-phosphorylation to the center of the protein. Mutational analysis demonstrated the importance of serine 315 in the hyper-phosphorylation process, while other serine residues tested had a minor affect. Live-cell imaging demonstrated that serine mutations in other regions of the protein affected the dynamics of hDcp1a association with the PB structure. Our work demonstrates the control of PB dynamics during the cell cycle via phosphorylation.

mRNP granules

“…For instance, microtubule depolymerizing drugs prevent assembly of large stress granules 39,40 and germ granules in early zebrafish embryos, 41 whereas P-bodies become larger, and less mobile. 42,43 Specific dynein and kinesin motor proteins also localize in stress granules, and appear to facilitate assembly and disassembly of stress granules, respectively. 40 Dynein proteins also increase P-body assembly under stress.…”

“…52,53 O-linked N-acetyl glucosamine, 53 poly (ADP) ribosylation, 54 acetylation, and ubiqutination 39 are also modifications present on specific stress granule components, which have been implicated in stress granule assembly, albeit the mechanisms are not entirely clear. Finally, the phosphorylation of several proteins, including G3BP, 29 TTP, 55 Dcp1, 42,56 Dcp2, 57 and 4E-T 58 alters their localization within, and/or the assembly of their respective granules. In addition, phosphorylation in the prion-like domain of FUS impairs hydrogel formation in vitro.…”

mRNA Decapping Enzyme 1a (Dcp1a)-induced Translational Arrest through Protein Kinase R (PKR) Activation Requires the N-terminal Enabled Vasodilator-stimulated Protein Homology 1 (EVH1) Domain

“…Previous studies have indicated roles for Dcp1a in P-body formation, maintenance, and regulation (6,32,33). Dcp1a in P-bodies constantly and quickly exchanges with Dcp1a in the cytoplasmic pool during fluorescence recovery after photobleaching analysis (34).…”

“…Interestingly, the EVH1 domain of Dcp1a is highly conserved, but the substrate specificity varies across species (42). Recent studies have looked at the status of Dcp1a and P-bodies during stages of the cell cycle and the importance of Dcp1a phosphorylation for its function (32,33,36). These studies have highlighted the importance of Dcp1a phosphorylation in several signaling pathways that affect P-body formation and maintenance.…”

“…In an effort to understand the importance of Dcp1a cleavage to P-body disruption and further elucidate Dcp1a biology, we utilized the well documented GFP-Dcp1a expression system (32,33,36). Previous studies utilizing the GFP-Dcp1a overexpression system have demonstrated that GFP-Dcp1a retains the same functions as endogenous Dcp1a (32,33,35,36) and can enter and function in active decapping complexes and P-bodies (35). Additionally, GFP-Dcp1a expression has been used to determine alterations in the phosphorylation status of Dcp1a throughout the cell cycle and in response to cellular stress (32,33).…”