Characterization of p96h2bk: immunoreaction with an anti-Erk(extracellular-signal-regulated kinase) peptide antibody and activity in Xenopus oocytes and eggs.
We have shown previously that oncogenic Ras induces cell cycle arrest in activated Xenopus egg extracts [Pan, Chen and Lin (1994) J. Biol. Chem. 269, 5968-5975]. The cell cycle arrest correlates with the stimulation of a protein kinase activity that phosphorylates histone H2b in vitro (designated p96(h2bk)) [Chen and Pan (1994) J. Biol. Chem. 269, 28034-28043]. We report here that p96(h2bk) is likely to be p96(ram), a protein of approx. 96 kDa that immunoreacts with a monoclonal antibody (Mk-1) raised against a synthetic peptide derived from a sequence highly conserved in Erk1/Erk2 (where Erk is extracellular-signal-regulated kinase). This is supported by two lines of evidence. First, activation/inactivation of p96(h2bk) correlates with upward/downward bandshifts of p96(ram) in polyacrylamide gels. Secondly, both p96(h2bk) and p96(ram) can be immunoprecipitated by antibody Mk-1. We also studied the activity of p96(h2bk)/p96(ram) in Xenopus oocytes and eggs. p96(h2bk)/p96(ram) was inactive in stage 6 oocytes, was active in unfertilized eggs, and became inactive again in eggs after fertilization. Since stage 6 oocytes are at G2-phase of the cell cycle, unfertilized eggs arrest at M-phase and eggs exit M-phase arrest after fertilization, the results thus indicate that p96(h2bk)/p96(ram) activity is cell cycle dependent. Moreover, microinjection of oncogenic Ras into fertilized eggs at the one-cell stage arrests the embryos at the two-cell stage, and this induced arrest is correlated with an inappropriate activation of p96(h2bk)/p96(ram). The data are consistent with the concept that inappropriate activation of p96(h2bk)/p96(ram) plays a role in the cell cycle arrest induced by oncogenic Ras.
Chen
D H
DH
Department of Surgery, Division of Neurosurgery, University of Kentucky Chandler Medical Center, 800 Rose Street, Lexington, KY 40536, USA.
Chen
C T
CT
Zhang
Y
Y
Liu
M A
MA
Campos-Gonzalez
R
R
Pan
B T
BT
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D008297:Male; D008970:Molecular Weight; D009865:Oocytes / Q000187:drug effects / Q000201:enzymology; D010063:Ovum / Q000187:drug effects / Q000201:enzymology*; D010446:Peptide Fragments / Q000276:immunology; D011374:Progesterone / Q000494:pharmacology; D006656:Protamine Kinase / Q000276:immunology*; D014981:Xenopus; D015053:Zygote / Q000187:drug effects / Q000201:enzymology; D018631:ras Proteins / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D011374:Progesterone; D006656:Protamine Kinase; C105680:histone h2b kinase; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D018631:ras Proteins