Kicked by Mos and tuned by MPF-the initiation of the MAPK cascade in Xenopus oocytes

Abstract

The mitogen-activated protein kinase (MAPK) cascade is a paradigmatic signaling cascade, which plays a crucial role in many aspects of cellular events. The main initiator of the cascade in Xenopus oocytes is the oncoprotein Mos. After activation of the cascade, Mos activity is stabilized by MAPK via a feedback loop. Mos concentration levels are, however, not controlled by MAPK alone. In this paper we show, by imposing either a sustained or a peaked activity of M-phase promoting factor (MPF) (Cdc2-cyclin B), how the latter regulates the dynamics of Mos. Our experiments are supported by a detailed kinetic model for the Mos-MPF-MAPK network, which takes into account the three different phosphorylation states of Mos and, as a consequence, allows us to determine the time evolution of Mos under control of MPF. Our work opens a path toward a more complete and biologically realistic quantitative understanding of the dynamic interdependence of Mos and MPF in Xenopus oocytes.

Figure 1. (A) Mos phosphorylations states.

Folded Mos (X) is at first inhibited by phosphorylation of residue Ser-105, located in a α-helix domain and promoting its association with CK2β. Phosphorylated on Ser-3, X, as well as X_a, are targeted for proteasomal degradation, presumably through their association with the anaphase promoting complex. Beside its role in protein stabilization, Ser-3 phosphorylation enhances catalytic activity of X_as toward Y/MEK. (See text for further discussion.) (B) Network structure of the MAPK cascade activation in Xenopus oocytes as studied in the present work. Molecules are indicated by their abbreviating symbols in the kinetic model, whereby X,Y,Z with their indices standing for the three corresponding phosphorylation states of Mos, MEK, and MAPK, respectively.

Figure 2. Bifurcation analysis of the Mos-MPF-MAPK ODE model.

(A) State diagram in the space of the kinetic parameters k₃, (Mos-MPF) and k₄ (Mos-MAPK). (B) State diagram in the space of variables [X_as; (MPF)] for the case of a bistable transition [region II in (B)]. (C) As (B) but for the irreversible case [region III in (B)].

Figure 3. Mos accumulates in the absence of MAPK following progesterone addition.

(A) Histogram depicts normalized values of Mos concentration at GVBD time in control oocytes treated with DMSO vehicle (0%,1%) and in U0126-treated oocytes (50 μM). Mos levels are, respectively, 1±0.22 and 0.83±0.27. (B) Cell by cell analysis by western blot for contents in Mos, MAPK, cyclin B2, phospho-tyrosine 15 Cdc2, and actin. Oocytes were taken at GVBD time for biochemical analysis.

Figure 4. Sustained accumulation of Mos follows egg-cytoplasm injection.

Donors and recipient were subjected to western blot analysis (A). (B) Network structure of the MAPK cascade activation in Xenopus oocytes: MPF is proposed to interact with S (dashed line); (C) normalized values for Mos from western blots, theoretical time evolution of the concentration of MPF (bold) and total active form of Mos (X_a+X_as). The concentration of MPF has been chosen in order to reach a constant maximal value of Mos as in the experiment. For further discussion see text.

Figure 5. Mos accumulation dynamics in the absence of the MAPK-driven feedback loop.

In the absence of MAPK activity, Mos transiently accumulates following metaphase II egg (donor) cytoplasm injection. Meiotic resumption was stimulated in presence of U0126 by 50 nl injection of metaphase arrested oocytes, rinsed of progesterone. Donors and recipient oocytes were subjected to western blot analysis (A) for their contents in Mos, Erk2, Rsk1, and cyclin B2. Neither Erk nor Rsk exhibits active profiles; PI, prophase I arrested oocytes; MII, Metaphase II arrested oocytes; Pg, progesterone; Cy, MPF-containing cytoplasm from MII oocytes. (B) Network structure of the MAPK cascade activation in Xenopus oocytes: the interaction of Z₃ with X_a is broken by U0126, a chemical inhibitor of MEK/Y (dashed lines). MPF activation is achieved in the absence of input as described in (A) and MPF is proposed to interact with S (dashed line). (C) Normalized values for Mos level are depicted for control and U0126-treated oocytes injected with MPF-containing cytoplasm. Numerically calculated time evolution of the concentration of MPF (bold), and total active form of Mos (X_a+X_as). The concentration of MPF follows an assumed χ²-shape leading to a profile of Mos qualitatively consistent with experiment. For further discussion see text.

Figure 6. Mos accumulation pattern following progesterone stimulation in presence of MEK-inhibitor U0216.

(A) Network structure of the MAPK cascade activation in Xenopus oocytes: Z₃-interaction with X_a is broken by U0126, chemical inhibitor of MEK∕Y (dashed lines). MPF activation and in are brought by hormonal stimulation. (B) Representative experiment (n=3 females). Dashed arrow: GVBD50, arrow: MPK activity peak. (C) Normalized values for Mos and numerically calculated time evolution of the concentration of MPF (bold) and total active form of Mos (X_a+X_as).

Figure 7. Supposed bimodal time evolution of the concentration of MPF and resulting total active form of Mos

(X_a+X_as). The resulting curve for Mos clearly shows a complex nonlinear variation with the input signal.