The proto-oncogene p120(Cbl) is a downstream substrate of the Hck protein-tyrosine kinase

Hematopoietic cell kinase (Hck) is a member of the Src-family of protein tyrosine kinases. We have found that upon enzymatic activation of Hck by the heavy metal mercuric chloride, there was a rapid increase in the levels of tyrosine phosphorylation of several proteins including the proto-oncogene p120(Cbl). Fibroblasts that are transformed with an activated allele of Hck exhibit constitutive Cbl phosphorylation. Upon Fcgamma receptor activation, a more physiologically relevant extracellular signal, Cbl is tyrosine phosphorylated and the Src-family selective inhibitor, PP1, can prevent this phosphorylation on Cbl. Hck phosphorylates Cbl in vitro and the interaction between Cbl and Hck is direct, requiring Hck's unique, SH3 and SH2 domains for optimal binding. Using a novel estrogen-regulated chimera of Hck we have shown a hormone-dependent association between Hck and Cbl in murine fibroblasts. This work suggests that Cbl serves as a key mediator of Hck induced signalling in hematopoietic cells.     

The RRASK motif in Xenopus cyclin B2 is required for the substrate recognition of Cdc25C by the cyclin B-Cdc2 complex

The FLRRXSK sequence is conserved in the second cyclin box fold of B-type cyclins. We show that this conserved sequence in Xenopus cyclin B2, termed the RRASK motif, is required for the substrate recognition by the cyclin B-Cdc2 complex of Cdc25C. Mutations to charged residues of the RRASK motif of cyclin B2 abolished its ability to activate Cdc2 kinase without affecting its capacity to bind to Cdc2. Cdc2 bound to the cyclin B2 RRASK mutant was not dephosphorylated by Cdc25C, and as a result, the complex was inactive. The cyclin B2 RRASK mutants can form a complex with the constitutively active Cdc2, but a resulting active complex did not phosphorylate a preferred substrate Cdc25C in vitro, although it can phosphorylate the non-specific substrate histone H1. The RRASK mutations prevented the interaction of Cdc25C with the cyclin B2-Cdc2 complex. Consistently, the RRASK mutants neither induced germinal vesicle breakdown in Xenopus oocyte maturation nor activated in vivo Cdc2 kinase during the cell cycle in mitotic extracts. These results suggest that the RRASK motif in Xenopus cyclin B2 plays an important role in defining the substrate specificity of the cyclin B-Cdc2 complex.     

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 Xenopus cdc2 protein is a component of MPF, a cytoplasmic regulator of mitosis

In Xenopus, a cytoplasmic agent known as MPF induces entry into mitosis. In fission yeast, genetic studies have shown that the cdc2 kinase regulates mitotic initiation. The 13 kd product of the suc1 gene interacts with the cdc2 kinase in yeast cells. We show that the yeast suc1 gene product (p13) is a potent inhibitor of MPF in cell-free extracts from Xenopus eggs. p13 appears to exert its antagonistic effect by binding directly to MPF. MPF activity is quantitatively depleted by chromatography on a p13 affinity column. Concomitantly, the Xenopus counterpart of the yeast cdc2 protein is adsorbed to the column. A 42 kd protein also binds specifically to the p13 affinity matrix. These findings suggest that the Xenopus cdc2 protein and the 42 kd protein are components of MPF.     

Degradation of the proto-oncogene product p39mos is not necessary for cyclin proteolysis and exit from meiotic metaphase: requirement for a Ca(2+)-calmodulin dependent event.

Exit from M phase, which requires cyclin degradation, is prevented from occurring in unfertilized eggs of vertebrates arrested at second meiotic metaphase due to a cytostatic factor recently identified as p39mos, the product of the proto-oncogene c-mos. Calpain can destroy both p39mos and cyclin in vitro in extracts prepared from metaphase-arrested Xenopus eggs, but only when free Ca2+ concentration is raised to the millimolar range. When free Ca2+ concentration is raised for only 30 s to the micromolar range, as occurs in physiological conditions after fertilization, cyclin degradation is induced, byt p39mos is not degraded. Cyclin proteolysis at micromolar free Ca2+, is not inhibited by calpastatin, and therefore does not involve calpain. A cyclin mutant modified in the destruction box is found to be resistant at micromolar, but not millimolar free Ca2+, suggesting that the ubiquitin pathway mediates cyclin degradation at micromolar Ca2+ concentration whereas calpain is involved at the millimolar level. A synthetic peptide which binds Ca(2+)-calmodulin with high affinity suppresses cyclin degradation at micromolar but not millimolar free Ca2+, and this only when it is present in the extract during the first 30 s after raising free Ca2+ concentration. The inhibition of the cyclin degradation pathway by the Ca(2+)-calmodulin binding peptide can be overcome by adding calmodulin. These results strongly suggest that a Ca(2+)-calmodulin process is required as an early event following fertilization to release the cyclin degradation pathway from inhibition in metaphase-arrested eggs. In contrast, p39mos degradation is not required.     

cdc2 is a component of the M phase-specific histone H1 kinase: evidence for identity with MPF

A so-called "growth-associated" or "M phase-specific" histone H1 kinase (H1K) has been described in a wide variety of eukaryotic cell types. In starfish oocytes, the hormone 1-methyladenine triggers synchronous meiotic divisions that are accompanied by a rapid 30-fold stimulation of H1K activity. We have substantially purified this activated enzyme and find that it is enriched for a protein of 34 kd. Quantitative immunoblotting of the column fractions with antibodies raised against p34, the product of the fission yeast cdc2 gene, revealed complete coelution of the H1K activity and a 34 kd anti-cdc2 cross-reactive protein. Starfish H1K also displayed the same apparent molecular weight, on a molecular sizing column, as the mitotically activated p13/p34/p62 protein kinase complex of HeLa cells. p13, the product of the fission yeast suc1+ gene, interacts tightly with p34 in yeast, Xenopus, and HeLa cells. H1K from starfish binds strongly to p13-Sepharose and the time course of 1-methyladenine-induced H1K activation, whether assayed in crude extract or on p13-Sepharose beads, is identical. These results indicate that a cdc2 homolog is a subunit of the M phase-specific H1K of starfish meiotic oocytes. Since this protein is also a subunit of the M-phase promoting factor (MPF) of Xenopus oocytes, we suggest that H1K and MPF are the same entity, and that histone H1 is likely to be one substrate of the pleiotropic MPF.     

MPF from starfish oocytes at first meiotic metaphase is a heterodimer containing one molecule of cdc2 and one molecule of cyclin B.

We have purified to near homogeneity the M-phase-specific protein kinase from starfish oocytes at first meiotic metaphase, using an improved procedure based on affinity chromatography on the immobilized yeast protein suc1. As already reported, this is identical to MPF, the cytoplasmic factor that controls entry of eukaryotic cells into M-phase. MPF is a complex formed by the stoichiometric association of a 34-kd polypeptide previously identified as cdc2 with a polypeptide that migrates with the same mobility as starfish cyclin in SDS-PAGE (apparent mol. wt 47 kd). A cDNA clone encoding starfish cyclin B has been isolated and its sequence determined. It contains a single open reading frame encoding a predicted 43 729-dalton protein. Partial microsequencing of the 47-kd polypeptide component of MPF allowed its identification as the starfish cyclin. Since the apparent mol. wt of native starfish MPF was found to be less than 100 kd, it is a heterodimer comprising one molecule of cdc2 and one molecule of cyclin B.     

Behavior of cyclin B and cyclin B-dependent kinase during starfish oocyte meiosis reinitiation: evidence for non-identity with MPF

We reconsider the notion of MPF initiation, which is generally viewed as the prerequisite production of a small cdc2-cyclin B kinase activity sufficient to trigger its own self-amplification. Using starfish oocytes as a tool, we show that H1 kinase activation is not explosive, but rather is a gradual process in both time and space. We show further that the production of even a large H1 kinase activity, either in MPF-microinjected or in hormone-stimulated oocytes, is not sufficient to trigger MPF amplification.     

The c-mos proto-oncogene protein kinase turns on and maintains the activity of MAP kinase, but not MPF, in cell-free extracts of Xenopus oocytes and eggs.

During studies of the activation and inactivation of the cyclin B-p34cdc2 protein kinase (MPF) in cell-free extracts of Xenopus oocytes and eggs, we found that a bacterially expressed fusion protein between the Escherichia coli maltose-binding protein and the Xenopus c-mos protein kinase (malE-mos) activated a 42 kDa MAP kinase. The activation of MAP kinase on addition of malE-mos was consistent, whereas the activation of MPF was variable and failed to occur in some oocyte extracts in which cyclin A or okadaic acid activated both MPF and MAP kinase. In cases when MPF activation was transient, MAP kinase activity declined after MPF activity was lost, and MAP kinase, but not MPF, could be maintained at a high level by the presence of malE-mos. When intact oocytes were treated with progesterone, however, the activation of MPF and MAP kinase occurred simultaneously, in contrast to the behaviour of extracts. These observations suggest that one role of c-mos may be to maintain high MAP kinase activity in meiosis. They also imply that the activation of MPF and MAP kinase in vivo are synchronous events that normally rely on an agent that has still to be identified.     

Cytohesin-associated scaffolding protein (CASP) is a substrate for granzyme B and ubiquitination

Natural killer (NK) cells are a sub-population of cytotoxic lymphocytes that can kill tumor or infected cells without prior exposure, by secreting the contents of preformed cytotoxic vesicles, containing perforin and granzymes, at the immune synapse. Cytohesin-associated scaffolding protein (CASP) is an adaptor molecule uniquely expressed in lymphocytes that forms complexes with both vesicle-initiating and sorting proteins, and has roles in NK cell migration, cytotoxicity, and cytokine secretion. In this study, we show that CASP contains a conserved granzyme B cleavage site that could modify its intracellular localization and interaction with sorting nexin 27. We also provide evidence that CASP is modified by ubiquitination. Both of these post-translational modifications could rapidly modify CASP function and highlight the dynamic regulatory mechanisms that direct its role in NK cell functions.     

The P gene product of hepatitis B virus is required as a structural component for genomic RNA encapsidation.

Encapsidation of the pregenomic RNA into nucleocapsids is a selective process which depends on specific RNA-protein interactions. The signal involved in the packaging of the hepatitis B virus (HBV) RNA pregenome was recently defined as a short sequence located near the 5' end of that molecule (Junker-Niepmann et al., EMBO J., in press), but it remained an open question which viral proteins are required. Using a genetic approach, we analyzed whether proteins derived from the HBV P gene play an important role in pregenome encapsidation. The results obtained with point mutations, deletions, and insertions scattered throughout the P gene clearly demonstrate that (i) a P gene product containing all functional domains is required both for the encapsidation of HBV pregenomic RNA and for packaging of nonviral RNAs fused to the HBV encapsidation signal, (ii) known enzymatic activities are not involved in the packaging reaction, suggesting that P protein is required as a structural component, and (iii) P protein acts primarily in cis, i.e., pregenomic RNAs from which P protein is synthesized are preferentially encapsidated.     

CPEB controls the cytoplasmic polyadenylation of cyclin, Cdk2 and c-mos mRNAs and is necessary for oocyte maturation in Xenopus.

Cytoplasmic polyadenylation is a key mechanism controlling maternal mRNA translation in early development. In most cases, mRNAs that undergo poly(A) elongation are translationally activated; those that undergo poly(A) shortening are deactivated. Poly(A) elongation is regulated by two cis-acting sequences in the 3'-untranslated region (UTR) of responding mRNAs, the polyadenylation hexanucleotide AAUAAA and the U-rich cytoplasmic polyadenylation element (CPE). Previously, we cloned and characterized the Xenopus oocyte CPE binding protein (CPEB), showing that it was essential for the cytoplasmic polyadenylation of B4 RNA. Here, we show that CPEB also binds the CPEs of G10, c-mos, cdk2, cyclins A1, B1 and B2 mRNAs. We find that CPEB is necessary for polyadenylation of these RNAs in egg extracts, suggesting that this protein is required for polyadenylation of most RNAs during oocyte maturation. Our data demonstrate that the complex timing and extent of polyadenylation are partially controlled by CPEB binding to multiple target sites in the 3' UTRs of responsive mRNAs. Finally, injection of CPEB antibody into oocytes not only inhibits polyadenylation in vivo, but also blocks progesterone-induced maturation. This is due to inhibition of polyadenylation and translation of c-mos mRNA, suggesting that CPEB is critical for early development.     

The human ubiquitin conjugating enzyme UBE2J2 (Ubc6) is a substrate for proteasomal degradation

The human Ube2J2 enzyme functions in the ubiquitination of proteins at the ER. Here we demonstrate that it, and a second ubiquitin conjugating (Ubc) enzyme Ube2G2, are unstable, and incubation of transfected cells with proteasome inhibitors increased steady-state protein levels. For Ube2J2, pharmacological induction of the unfolded protein response (UPR) did not significantly alter ectopic protein levels, however the effect of proteasomal inhibition was abolished if the enzyme was inactivated or truncated to disrupt its ER-localization. These results suggest for the first time that the steady state expression of Ubcs’ may be important in regulating the degradation of ER proteins in mammalian cells.     

Requirement of cyclin B2, but not cyclin B1, for bipolar spindle formation in frog (Rana japonica) oocytes

Cyclin B, the regulatory subunit of maturation-promoting factor (MPF), comprises several subtypes that are presumed to confer different functions on MPF although no direct evidence has been provided to date. To clarify the difference in the roles of cyclins B1 and B2, we used frog (Rana japonica) oocytes in which MPF is formed only after progesterone stimulation because it is possible to produce oocytes containing either cyclin B1-MPF or cyclin B2-MPF by antisense RNA-mediated translational inhibition of each mRNA. Using this advantage, we investigated the functions of cyclins B1 and B2 and obtained the following results: (a) oocytes synthesizing cyclin B2-MPF underwent meiosis I and II with formation of a bipolar spindle at each metaphase; (b) oocytes synthesizing cyclin B1-MPF formed a monopolar spindle at metaphase I and extruded an abnormal polar body; and (c) both oocytes underwent germinal vesicle breakdown (GVBD) and chromosome condensation. Immunocytochemical observations also revealed continuous localization of cyclin B2 on the spindle during meiosis. These results provide evidence of the requirement of cyclin B2, but not cyclin B1, for organizing the bipolar spindle, though either cyclin B1 or B2 is redundant for inducing GVBD and chromosome condensation.