Identification of G2/M targets for the MAP kinase pathway by functional proteomics.
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.
Roberts
Elisabeth C
EC
Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder 80309-0215, USA.
Hammond
Kasey
K
Traish
Abdulmaged M
AM
Resing
Katheryn A
KA
Ahn
Natalie G
NG
D000595:Amino Acid Sequence; D002453:Cell Cycle / Q000502:physiology*; D004268:DNA-Binding Proteins; D015180:Electrophoresis, Gel, Two-Dimensional; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D020935:MAP Kinase Signaling System / Q000502:physiology*; D008969:Molecular Sequence Data; D034521:Nuclear Matrix-Associated Proteins / Q000378:metabolism; D050810:Octamer Transcription Factors / Q000378:metabolism; D020652:Peptide Elongation Factor 2 / Q000378:metabolism; D010766:Phosphorylation; D040901:Proteomics; D016601:RNA-Binding Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D004268:DNA-Binding Proteins; C083214:NONO protein, human; D034521:Nuclear Matrix-Associated Proteins; D050810:Octamer Transcription Factors; D020652:Peptide Elongation Factor 2; D016601:RNA-Binding Proteins; D048049:Extracellular Signal-Regulated MAP Kinases