ABSTRACT
In unfertilized Xenopus eggs, the p42 mitogen activated protein kinase (p42MAPK) pathway is known to maintain cell cycle arrest at metaphase of meiosis II. However, constitutive activation of p42MAPK in post-meiotic, cycling Xenopus egg extracts can lead to either a G2 or M-phase arrest of the cell cycle, depending on the timing of p42MAPK activation. Here, we examined the molecular mechanism by which activation of the p42MAPK pathway during interphase leads to cell cycle arrest in G2. When either a recombinant wild type Cdc25C(WT) or a mutated form of Cdc25C, in which serine 287 was replaced by an alanine (S287A), was added to cycling egg extracts, S287A accelerated entry into M-phase. Furthermore, the addition of S287A overcame the G2 arrest caused by p42MAPK, driving the extract into M-phase. p90Rsk a kinase that is the target of p42MAPK, was phosphorylated and activated (pp90Rsk) in the G2-arrested egg extracts, and was able to phosphorylate WT but not S287A in vitro. 14-3-3 proteins were associated with endogenous Cdc25C in G2-arrested extracts. Cdc25C(WT) that had been phosphorylated by pp90(Rsk) bound 14-3-3zeta, whereas S287A could not. These data suggest that the link between the p42MAPK signaling pathway and Cdc25C involves the activation of pp90Rsk and its phosphorylation of Cdc25C at S287, causing the binding of 14-3-3 proteins. We propose that the binding of 14-3-3 proteins to pp90Rsk phosphorylated-Cdc25C results in a G2 arrest in a manner similar to the cell cycle delays induced by differentiation signals that occur later in embryonic development.
Subject(s)
Cell Cycle Proteins/metabolism , Cell Cycle , G2 Phase , Oocytes/cytology , Ribosomal Protein S6 Kinases, 90-kDa/metabolism , cdc25 Phosphatases/metabolism , 14-3-3 Proteins/physiology , Alanine/analysis , Animals , Cell Cycle/physiology , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Cell Division/physiology , Cell Extracts , Enzyme Activation/genetics , Female , Gene Expression Regulation, Enzymologic , Immunoprecipitation , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 1/physiology , Oocytes/physiology , Phosphorylation , Protein Binding , Recombinant Proteins/metabolism , Serine/analysis , Xenopus , Xenopus Proteins/chemistry , Xenopus Proteins/genetics , Xenopus Proteins/physiology , cdc25 Phosphatases/chemistry , cdc25 Phosphatases/geneticsABSTRACT
The receptor-like protein tyrosine phosphatase CD45 is essential for TCR signal transduction. Substrates of CD45 include the protein tyrosine kinases p56(lck) and p59(fyn), both of which have been shown to be enriched in detergent-insoluble microdomains. Here we find that there is a cholesterol-dependent association between CD45 and the raft-associated protein linker for activation of T cells, suggesting that CD45 and linker for activation of T cells may colocalize in lipid rafts. Consistent with this observation, we find that approximately 5% of total CD45 can be detected in Triton X-100-insoluble buoyant fractions of sucrose gradients, demonstrating that CD45 is not excluded from lipid rafts. Upon stimulation of T cells with anti-CD3, there is a reduction in the amount of CD45 found associating with lipid rafts. Our data suggest that CD45 is present in lipid rafts in T cells before activation, perhaps to activate raft-associated p56(lck), allowing membrane-proximal signaling events to proceed. Furthermore, the reduction in CD45 content of lipid rafts after CD3 stimulation may serve to limit the amounts of activated p56(lck) in rafts and thus possibly the duration of T cell responses.
