Joan V. Ruderman

cdc2 protein kinase is complexed with both cyclin A and B: Evidence for proteolytic inactivation of MPF

In the clam, Spisula, two previously described proteins known as cyclin A and B display the unusual property of selective proteolytic degradation at the end of each mitosis. We show here that clam oocytes and embryos contain a cdc2 protein kinase. This protein kinase is a component of the M phase promoting factor (MPF) in frog eggs and the M phase-specific histone H1 kinase in starfish. Clam cdc2 is found in association with both cyclin A and B, probably not as a trimolecular association, but as separate cdc2/cyclin A and cdc2/cyclin B complexes. Clam cdc2 and the associated cyclins bind to p13suc1-Sepharose. The p13-bound complex, and also anti-cyclin A or B immunoprecipitates, each display cell cycle-dependent histone H1 kinase activity. We suggest that in addition to the cdc2 protein kinase, the cyclins are further components of the M phase promoting factor and that cyclin proteolysis provides the mechanism of MPF inactivation and thus exit from mitosis.

The clam embryo protein cyclin A induces entry into M phase and the resumption of meiosis in Xenopus oocytes

Fertilized clam embryos synthesize several new cell-cycle-related proteins. The cloned cDNA and derived amino acid sequences of one of these, cyclin A, are presented here. Immunoblots with an anti-cyclin A antibody reveal that cyclin A is undetectable in oocytes, appears within 15 min of fertilization, and is destroyed near the end of each meiosis and mitosis. We directly tested the ability of cyclin A to induce M phase by injecting SP6 cyclin A mRNA into Xenopus oocytes, which are arrested at the G2/M border of first meiosis. The injected mRNA was translated, with the result that the Xenopus oocytes entered meiosis. These findings indicate that the rise in cyclin A plays a direct and natural role in driving cells into M phase.

A Ran signalling pathway mediated by the mitotic kinase Aurora A in spindle assembly

The activated form of Ran (Ran-GTP) stimulates spindle assembly in Xenopus laevis egg extracts, presumably by releasing spindle assembly factors, such as TPX2 (target protein for Xenopus kinesin-like protein 2) and NuMA (nuclear-mitotic apparatus protein) from the inhibitory binding of importin-α and -β. We report here that Ran-GTP stimulates the interaction between TPX2 and the Xenopus Aurora A kinase, Eg2. This interaction causes TPX2 to stimulate both the phosphorylation and the kinase activity of Eg2 in a microtubule-dependent manner. We show that TPX2 and microtubules promote phosphorylation of Eg2 by preventing phosphatase I (PPI)-induced dephosphorylation. Activation of Eg2 by TPX2 and microtubules is inhibited by importin-α and -β, although this inhibition is overcome by Ran-GTP both in the egg extracts and in vitro with purified proteins. As the phosphorylation of Eg2 stimulated by the Ran-GTP–TPX2 pathway is essential for spindle assembly, we hypothesize that the Ran-GTP gradient established by the condensed chromosomes is translated into the Aurora A kinase gradient on the microtubules to regulate spindle assembly and dynamics.

Selective translation of mRNA controls the pattern of protein synthesis during early development of the surf clam, Spisula solidissima.

Abstract Striking changes in the pattern of protein synthesis occur shortly after the fertilization of Spisula solidissima oocytes. These changes include a strong reduction in the synthesis of prominent oocyte-specific proteins and a large increase in the synthesis of at least three proteins whose labeling dominates the pattern of protein synthesis in early embryos. Several independent lines of evidence suggest that these changes are modulated at the translational level: 1.—Oocytes and embryos contain identical sets of mRNA, as measured by cell-free translation of phenol-extracted RNA from the two developmental stages. 2.—Different sets of these messages are associated with ribosomes at the two stages. 3.—Cell-free translation of mixtures of the unextracted 12,000 × g supernatants of oocyte or embryo homogenates with reticulocyte lysate gives patterns of protein synthesis that accurately resemble the patterns of synthesis from intact cells of the cognate stage, despite the fact that these homogenates contain identical sets of mRNA. These results provide strong evidence that the alterations in the pattern of protein synthesis at fertilization are due to stage-specific utilization of different subsets of messenger RNA from a common maternal mRNA population at the two stages of development. The fact that phenol extraction of these mRNAs renders them all equally available for translation in the reticulocyte lysate implies that discrimination is achieved by selective repression of availability by some phenol-soluble component of the cells.

Identification of phosphorylated residues that affect the activity of the mitotic kinase Aurora-A

The activity of the kinase Aurora-A (Aur-A) peaks during mitosis and depends on phosphorylation by one or more unknown kinases. Mitotic phosphorylation sites were mapped by mass spec sequencing of recombinant Aur-A protein that had been activated by incubation in extracts of metaphase-arrested Xenopus eggs. Three sites were identified: serine 53 (Ser-53), threonine 295 (Thr-295), and serine 349 (Ser-349), which are equivalent to Ser-51, Thr-288, and Ser-342, respectively, in human Aur-A. To ask how phosphorylation of these residues might affect kinase activity, each was mutated to either alanine or aspartic acid, and the recombinant proteins were then tested for their ability to be activated by M phase extract. Mutation of Thr-295, which resides in the activation loop of the kinase, to either alanine or aspartic acid abolished activity. The S349A mutant had slightly reduced activity, indicating that phosphorylation is not required for activity. The S349D mutation completely blocked activation, suggesting that Ser-349 is important for either the structure or regulation of Aur-A. Finally, like human Aur-A, overexpression of Xenopus Aur-A transformed NIH 3T3 cells and led to tumors in nude mice. These results provide further evidence that Xenopus Aur-A is a functional ortholog of human Aur-A and, along with the recently described crystal structure of human Aur-A, should help in future studies of the mechanisms that regulate Aur-A activity during mitotic progression.

G2 arrest in Xenopus oocytes depends on phosphorylation of cdc25 by protein kinase A

Xenopus oocytes, which are arrested in G2 of meiosis I, contain complexes of cyclin B–cdc2 (M phase-promoting factor) that are kept repressed by inhibitory phosphorylations on cdc2 at Thr-14 and Tyr-15. Progesterone induces a cytoplasmic signaling pathway that leads to activation of cdc25, the phosphatase that removes these phosphorylations, catalyzing entry into M phase. It has been known for 25 years that high levels of cAMP and protein kinase A (PKA) are required to maintain the G2 arrest and that a drop in PKA activity is required for M phase-promoting factor activation, but no physiological targets of PKA have been identified. We present evidence that cdc25 is a critical target of PKA. (i) In vitro, cdc25 Ser-287 serves as a major site of phosphorylation by PKA, resulting in sequestration by 14-3-3. (ii) Endogenous cdc25 is phosphorylated on Ser-287 in oocytes and dephosphorylated in response to progesterone just before cdc2 dephosphorylation and M-phase entry. (iii) High PKA activity maintains phosphorylation of Ser-287 in vivo, whereas inhibition of PKA by its heat-stable inhibitor (PKI) induces dephosphorylation of Ser-287. (iv) Overexpression of mutant cdc25 (S287A) bypasses the ability of PKA to maintain oocytes in G2 arrest. These findings argue that cdc25 is a physiologically relevant target of PKA in oocytes. In the early embryonic cell cycles, Ser-287 is phosphorylated during interphase and dephosphorylated just before cdc2 activation and mitotic entry. Thus, in addition to its role in checkpoint arrest, cdc25 Ser-287 serves as a site for regulation during normal, unperturbed cell cycles.

Quantitative NMR analysis of the protein G B1 domain in Xenopus laevis egg extracts and intact oocytes

We introduce a eukaryotic cellular system, the Xenopus laevis oocyte, for in-cell NMR analyses of biomolecules at high resolution and delineate the experimental reference conditions for successful implementations of in vivo NMR measurements in this cell type. This approach enables quantitative NMR experiments at defined intracellular concentrations of exogenous proteins, which is exemplified by the description of in-cell NMR properties of the protein G B1 domain (GB1). Additional experiments in Xenopus egg extracts and artificially crowded in vitro solutions suggest that for this biologically inert protein domain, intracellular viscosity and macromolecular crowding dictate its in vivo behavior. These contributions appear particularly pronounced for protein regions with high degrees of internal mobility in the pure state. We also evaluate the experimental limitations of this method and discuss potential applications toward the in situ structural characterization of eukaryotic cellular activities.

Activation of p42 MAP kinase and the release of oocytes from cell cycle arrest.

Clam oocytes are arrested naturally at the G2/M border in meiosis and contain an inactive 42 kDa ERK/MAP kinase, p42MAPK. Following fertilization, p42MAPK is rapidly phosphorylated on tyrosine residues and concomitantly activated. Both tyrosine phosphorylation and activation of p42MAPK begin within 2–3 min of fertilization, peak at approximately 15 min, then rapidly decline and disappear around the end of meiosis I. Neither the tyrosine phosphorylated form of p42MAPK nor p42MAPK activity reappears during meiosis II or the succeeding mitotic cell cycles. High doses of molybdate, a potent PTPase inhibitor, block the phosphorylation of p42MAPK and entry into the cell cycle. Lower doses of molybdate delay both p42MAPK phosphorylation and the release from cell cycle arrest, but once cells have re‐entered the cell cycle, they continue with near‐normal timing. These results argue that the transient activation of p42MAPK at fertilization is a one‐time event linked to release from cell cycle arrest. In trying to reconcile this one‐time activation of p42MAPK in clam embryos with the recurring, M‐phase specific activation of MBP/MAP kinases reported in other systems, we show that cdc2 kinase contributes a major portion of the MBP kinase activity in mitotic extracts. Furthermore, a small fraction of p42MAPK and other related kinases are present in p13suc1‐bound material, cautioning against the use of p13suc1 beads for experiments where, in addition to cdc2, the unaccounted presence of other kinase activities could be misleading.

The kinase Eg2 is a component of the Xenopus oocyte progesterone-activated signaling pathway

Quiescent Xenopus oocytes are activated by progesterone, which binds to an unidentified surface‐associated receptor. Progesterone activates a poorly understood signaling pathway that results in the translational activation of mRNA encoding Mos, a MAP kinase kinase kinase necessary for the activation of MAP kinase and MPF, the resumption of meiosis, and maturation of the oocyte into the sperm‐responsive egg. We have designed a screen to identify early signaling proteins based on the premise that some of these proteins would be phosphorylated or otherwise modified within minutes of progesterone addition. This screen has revealed Eg2, a Ser/Thr kinase. We find that Eg2 is phosphorylated soon after progesterone stimulation and provide evidence that it functions in the signaling pathway. Overexpression of Eg2 via mRNA microinjection shortens the time between progesterone stimulation and the appearance of new Mos protein, accelerates activation of MAP kinase and advances entry into the meiotic cell cycle. Finally, overexpression of Eg2 dramatically reduces the concentration of progesterone needed to trigger oocyte activation. These results argue that the kinase Eg2 is a component of the progesterone‐activated signaling pathway that releases frog oocytes from cell cycle arrest.

Sequence-specific adenylations and deadenylations accompany changes in the translation of maternal messenger RNA after fertilization of Spisula oocytes

A dramatic change in the pattern of protein synthesis occurs within ten minutes after fertilization of Spisula oocytes. This change is regulated entirely at the translational level. We have used DNA clones complementary to five translationally regulated messenger RNAs to follow shifts in mRNA utilization at fertilization and to characterize alterations in mRNA structure that accompany switches in translational activity in vivo. Four of the mRNAs studied are translationally inactive in the oocyte. After fertilization two of these mRNAs are completely recruited onto polysomes, and two are partially recruited. All four of these mRNAs have very short poly(A) tracts in the oocyte; after fertilization the poly(A) tails lengthen considerably. In contrast, a fifth mRNA, that encoding alpha-tubulin mRNA, is translated very efficiently in the oocyte and is rapidly lost from polysomes after fertilization. Essentially all alpha-tubulin mRNA in the oocyte is poly(A)+ and a large portion of this mRNA undergoes complete deadenylation after fertilization. These results reveal a striking relationship between changes in adenylation and translational activity in vivo. This correlation is not perfect, however. Evidence for and against a direct role for polyadenylation in regulating these translational changes is discussed. Changes in poly(A) tails are the only alterations in mRNA sizes that we have been able to detect. This indicates that, at least for the mRNAs studied here, translational activation is not due to extensive processing of larger translationally incompetent precursors. We have also isolated several complementary DNA clones to RNAs encoded by the mitochondrial genome. Surprisingly, the poly(A) tracts of at least two of the mitochondrial RNAs also lengthen in response to fertilization.