Identification of G2/M targets for the MAP kinase pathway by functional proteomics

Abstract

Although the importance of the extracellular signal-regulated kinase (ERK) pathway in regulating the transition from G1 to S has been extensively studied, its role during the G2/M transition is less well understood. Previous reports have shown that inhibition of the ERK pathway in mammalian cells delays entry as well as progression through mitosis, suggesting the existence of molecular targets of this pathway in M phase. In this report we employed 2-DE and MS to survey proteins and PTMs in the presence versus absence of MKK1/2 inhibitor. Targets of the ERK pathway in G2/M were identified as elongation factor 2 (EF2) and nuclear matrix protein, 55 kDa (Nmt55). Phosphorylation of each protein increased under conditions of ERK pathway inhibition, suggesting indirect control of these targets; regulation of EF2 was ascribed to phosphorylation and inactivation of upstream EF2 kinase, whereas regulation of Nmt55 was ascribed to a delay in normal mitotic phosphorylation and dephosphorylation. 2-DE Western blots probed using anti-phospho-Thr-Pro antibody demonstrated that the effect of ERK inhibition is not to delay the onset of phosphorylation controlled by cdc2 and other mitotic kinases, but rather to regulate a small subset of targets in M phase in a nonoverlapping manner with cdc2.

Analysis of Viral and Cellular Factors Influencing Herpesvirus-Induced Nuclear Envelope Breakdown

“…The known substrate for MEK1/2 is ERK1/2 (7). The role of ERK activity during G 2 /M transition is not well understood, but ERK seems to function through a complex and indirect role on a small subset of G 2 /M phosphoproteins (55), and the downstream effectors of ERK, which are required for mitosis progression, remain to be determined (2). However, a specific inhibitor of ERK1/2 (FR180204) had no influence on the titers of either the passaged mutants or wild-type PrV.…”

The role of protein synthesis in cell cycling and cancer

“…The mTOR pathway has been extensively studied by Proud and co‐workers with a recent review providing an excellent summary (Proud, 2007). The contribution of the MKK pathway to EF2 kinase phosphorylation and the explicit role this plays in cell cycle has recently been examined by Roberts and co‐workers who demonstrate that phosphorylation of Ser366 rises quickly upon G1 entry in a synchronized cell population in an MKK‐dependent manner (Roberts et al., 2006).…”

Unbiased Functional Proteomics Strategy for Protein Kinase Inhibitor Validation and Identification ofbona fideProtein Kinase Substrates: Application to Identification of EEF1D as a Substrate for CK2

“…We employed two-dimensional electrophoresis to identify proteins exhibiting diminished phosphorylation in cells treated with CK2 inhibitors based on its capacity to fractionate thousands of individual protein variants, including separation of different phosphorylated forms of individual proteins, and its demonstrated ability to identify substrates for protein kinases such as MAP kinase. (50) To extend these studies, we generated inhibitor-resistant mutants of CK2(15) to evaluate whether the identified proteins are indeed direct substrates for CK2. Utilizing these strategies, we identified EEF1D, a translational elongation factor implicated as a potential prognostic indicator in cancer (including medulloblastoma(51) and esophageal carcinoma(52)) as a bona fide cellular target of CK2.…”