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A Xenopus 45 kDa protein has been identified as an immediate upstream factor sufficient for full activation of MAP kinase, and is shown to be capable of undergoing autophosphorylation on serine, threonine and tyrosine residues. In this study, we show that purified 45 kDa protein can phosphorylate a kinase‐negative mutant of Xenopus MAP kinase on tyrosine and threonine residues, suggesting that the 45 kDa protein functions as a MAP kinase kinase to activate MAP kinase. We then report the cloning and sequencing of a full‐length cDNA encoding this 45 kDa MAP kinase kinase, and show that it is highly homologous to four protein kinases in fission and budding yeasts: byr1, wis1, PBS2 and STE7. These yeast kinases are therefore suggested to function as a direct upstream activator for a presumed MAP kinase homolog in each signal transduction pathway involved in the regulation of cell cycle progression or cellular responses to extracellular signals. Finally, we report bacterial expression of recombinant MAP kinase kinase that can be phosphorylated and activated by Xenopus egg extracts.

MAP kinase is activated and phosphorylated during M phase of the Xenopus oocyte cell cycle, and induces the interphase‐M phase transition of microtubule dynamics in vitro. We have carried out molecular cloning of Xenopus M phase MAP kinase and report its entire amino acid sequence. There is no marked change in the MAP kinase mRNA level during the cell cycle. Moreover, studies with an anti‐MAP kinase antiserum indicate that MAP kinase activity may be regulated posttranslationally, most likely by phosphorylation. We show that MAP kinase can be activated by microinjection of MPF into immature oocytes or by adding MPF to cell‐free extracts of interphase eggs. These results suggest that MAP kinase functions as an intermediate between MPF and the interphase‐M phase transition of microtubule organization.

An activity that severs stable microtubules has previously been detected in M phase extracts, but not in interphase extracts, of Xenopus eggs. We show that incubation of interphase extracts with purified MPF rapidly increases the microtubule‐severing activity. We then report the identification and purification of a novel protein factor responsible for this MPF‐dependent microtubule‐severing activity. The purified microtubule‐severing factor is a homo‐oligomeric protein composed of 56 kDa polypeptide subunits. These subunits appear to assemble into a pentagonal loop, forming a doughnut‐shaped molecule whose overall contours resemble a flattened ball. The microtubule‐severing activity of the purified factor does not require ATP or divalent cations, and is inhibited by monomeric tubulin. The purified factor is capable of binding to both monomeric tubulin and microtubules. This factor is thus a novel kind of microtubule‐binding protein in both structure and function, and may play an important role in the cell cycle‐dependent change in microtubule organization.

MAP kinase is thought to play a pivotal role not only in the growth factor‐stimulated signalling pathway but also in the M phase phosphorylation cascade downstream of MPF. MAP kinase is fully active only when both tyrosine and threonine/serine residues are phosphorylated. We have now identified and purified a Xenopus MAP kinase activator from mature oocytes that is able to induce activation and phosphorylation on tyrosine and threonine/serine residues of an inactive form of Xenopus MAP kinase. The Xenopus MAP kinase activator itself is a 45 kDa phosphoprotein and is inactivated by protein phosphatase 2A treatment in vitro. Microinjection of the purified activator into immature oocytes results in immediate activation of MAP kinase. Further experiments using microinjection as well as cell free extracts have shown that Xenopus MAP kinase activator is an intermediate between MPF and MAP kinase. Thus, MAP kinase activator plays a key role in the phosphorylation cascade.

Mitogen‐activated protein kinase (MAPK) is activated by MAPK kinase (MAPKK) in a variety of signaling pathways. This kinase cascade has been shown to function in cell proliferation and differentiation, but its role in early vertebrate development remains to be investigated. During early vertebrate embryogenesis, the induction and patterning of mesoderm are thought to be determined by signals from intercellular factors such as members of the fibroblast growth factor (FGF) family and members of the transforming growth factor‐beta family. Here we show that the microinjection of either mRNA encoding a constitutively active mutant of MAPKK or mRNA encoding a constitutively active form of STE11, a MAPKK kinase, leads to the induction of mesoderm in ectodermal explants from Xenopus embryos. Moreover, the expression of MAPK phosphatase‐1 (MKP‐1, also called CL100) blocks the growth factor‐stimulated mesoderm induction. Furthermore, injection of CL100 mRNA into two‐cell stage embryos causes severe defects in gastrulation and posterior development. The effects induced by CL100 can be rescued by co‐injection of wild‐type MAPK mRNA. Thus, the MAPK cascade may play a crucial role in early vertebrate embryogenesis, especially during mesoderm induction.

Xenopus MAP kinase activator, a 45 kDa protein, has been shown to function as a direct upstream factor sufficient for full activation and both tyrosine and serine/threonine phosphorylation of inactive MAP kinase. We have now shown by using an anti‐MAP kinase activator antiserum that MAP kinase activator is ubiquitous in tissues and is regulated post‐translationally. Activation of MAP kinase activator is correlated precisely with its threonine phosphorylation during the oocyte maturation process. It is a key question whether MAP kinase activator is a kinase or not. We have shown that Xenopus MAP kinase activator purified from mature oocytes is capable of undergoing autophosphorylation on serine, threonine and tyrosine residues. Dephosphorylation of purified activator by protein phosphatase 2A treatment inactivates its autophosphorylation activity as well as its activator activity. Thus, Xenopus MAP kinase activator is a protein kinase with specificity for both serine/threonine and tyrosine. Partial protein sequencing of purified activator indicates that it contains a sequence homologous to kinase subdomains VI and VII of two yeast protein kinases, STE7 and byrl.

The interphase‐M phase transition of microtubule dynamics is thought to be induced by phosphorylation reactions mediated by MPF and by MAP kinase functioning downstream of MPF. We have now identified and purified from Xenopus eggs a major microtubule‐associated protein, p220, that may be a target protein for these two M phase‐activated kinases. p220, when purified from interphase cells, potently bound to microtubules and stimulated tubulin polymerization, whereas p220 purified from M phase cells showed little or no such activities. Cell staining with a monoclonal anti‐p220 antibody revealed that p220 is localized on cytoplasmic microtubule networks during interphase, while it is distributed rather diffusely throughout the cell during M phase. We have further found that p220 is phosphorylated specifically in M phase. Moreover, p220 purified from interphase cells served as a good substrate for MAP kinase and MPF in vitro, and two‐dimensional phosphopeptide mapping pattern of the p220 phosphorylated in vitro was very similar to that of p220 phosphorylated at M phase in vivo. These results suggest that the drastic change in p220 activity during the transition from interphase to M phase may be induced by its phosphorylation in M phase probably catalyzed by MAP kinase and MPF.

MAP kinase kinase (MAPKK) has been identified as a protein factor that can induce phosphorylation and activation of inactive MAP kinase in vitro. In this study, we produced an anti‐Xenopus MAPKK antibody that can specifically inhibit Xenopus MAPKK activity in vitro. Microinjection of this antibody into immature oocytes prevented progesterone‐induced MAP kinase activation. Moreover, progesterone‐induced histone H1 kinase activation and germinal vesicle breakdown (GVBD) were inhibited in the oocytes injected previously with this antibody. Furthermore, when a bacterially expressed Mos was introduced into immature oocytes, Mos‐induced MAP kinase activation and GVBD were blocked in the oocytes injected with the anti‐MAPKK antibody. These results show that MAPKK is responsible for the activation of MAP kinase in vivo and that the MAPKK/MAP kinase cascade plays a pivotal role in the MPF activation during the oocyte maturation process.