The proliferation and differentiation of cells of many lineages are regulated by secreted proteins known as cytokines. Cytokines exert their biological effect through binding to cell-surface receptors that are associated with one or more members of the JAK family of cytoplasmic tyrosine kinases. Cytokine-induced receptor dimerization leads to the activation of JAKs, rapid tyrosine-phosphorylation of the cytoplasmic domains, and subsequent recruitment of various signalling proteins, including members of the STAT family of transcription factors, to the receptor complex. Using the yeast two-hybrid system, we have now isolated a new SH2-domain-containing protein, JAB, which is a JAK-binding protein that interacts with the Jak2 tyrosine-kinase JH1 domain. JAB is structurally related to CIS, a cytokine-inducible SH2 protein. Interaction of JAB with Jak1, Jak2 or Jak3 markedly reduces their tyrosine-kinase activity and suppresses the tyrosine-phosphorylation and activation of STATs. JAB and CIS appear to function as negative regulators in the JAK signalling pathway.

Cyclin E was first identified by screening human cDNA libraries for genes that would complement G 1 cyclin mutations in Saccharomyces cerevisiae and has subsequently been found to have specific biochemical and physiological properties that are consistent with it performing a G 1 function in mammalian cells. Most significantly, the cyclin E-Cdk2 complex is maximally active at the G 1 /S transition, and overexpression of cyclin E decreases the time it takes the cell to complete G 1 and enter S phase. We have now found that mammalian cells express two forms of cyclin E protein which differ from each other by the presence or absence of a 15-amino-acid amino-terminal domain. These proteins are encoded by alternatively spliced mRNAs and are localized to the nucleus during late G 1 and early S phase. Fibroblasts engineered to constitutively overexpress either form of cyclin E showed elevated cyclin E-dependent kinase activity and a shortened G 1 phase of the cell cycle. The overexpressed cyclin E protein was detected in the nucleus during all cell cycle phases, including G 0 . Although the cyclin E protein could be overexpressed in quiescent cells, the cyclin E-Cdk2 complex was inactive. It was not activated until 6 to 8 h after readdition of serum, 4 h earlier than the endogenous cyclin E-Cdk2. This premature activation of cyclin E-Cdk2 was consistent with the extent of G 1 shortening caused by cyclin E overexpression. Microinjection of affinity-purified anti-cyclin E antibodies during G 1 inhibited entry into S phase, whereas microinjection performed near the G 1 /S transition was ineffective. These results demonstrate that cyclin E is necessary for entry into S phase. Moreover, we found that cyclin E, in contrast to cyclin D1, was required for the G 1 /S transition even in cells lacking retinoblastoma protein function. Therefore, cyclins E and D1 control two different transitions within the human cell cycle.Cyclins and their catalytic subunits, the cyclin-dependent kinases (Cdks), play key roles in the regulation of eukaryotic cell cycle events (see references 10 and 82 for reviews). Cyclins are believed to determine the subcellular localization, substrate specificity, interaction with upstream regulatory proteins, and timing of activation of Cdks.Cell cycle control mechanisms have been highly conserved throughout evolution (see reference 59 for a review). Cyclins were originally discovered in marine invertebrates. In clam and sea urchin embryos, A-and B-type cyclins were found to accumulate to high levels during interphase before they were abruptly degraded during mitosis (17). Control of the cell cycle by these cyclins was demonstrated when injection of clam cyclin A mRNA was able to advance G 2 -arrested Xenopus oocytes into meiotic metaphase (89). Furthermore, after destruction of all mRNA in Xenopus egg extracts, readdition of sea urchin cyclin B message was sufficient to drive the extract into mitosis (56).In mammalian cells, several cyclins have been identified and classified according to their sequence simil...

Eukaryotic cells become committed to proliferate during the G1 phase of the cell cycle. In budding yeast, commitment occurs when the catalytic subunit of a protein kinase, encoded by the CDC28 gene (the homolog of the fission yeast cdc2+ gene), binds to a positively acting regulatory subunit, a cyclin. Related kinases are also required for progression through the G1 phase in higher eukaryotes. The role of cyclins in controlling G1 progression in mammalian cells was tested by construction of fibroblasts that constitutively overexpress human cyclin E. This was found to shorten the duration of G1, decrease cell size, and diminish the serum requirement for the transition from G1 to S phase. These observations show that cyclin levels can be rate-limiting for G1 progression in mammalian cells and suggest that cyclin synthesis may be the target of physiological signals that control cell proliferation.

Abstract. Growth factors and cell anchorage jointly regulate transit through G1 in almost all cell types, but the cell cycle basis for this combined requirement remains largely uncharacterized. We show here that cell adhesion and growth factors jointly regulate the cyclin D1-and E-dependent kinases. Adhesion to substratum regulates both the induction and translation of cyclin D1 mRNA. Nonadherent cells fail to phosphorylate the retinoblastoma protein (Rb), and enforced expression of cyclin D1 rescues Rb phosphorylation and entry into S phase when G1 cells are cultured in the absence of substratum. Nonadherent cells also fail to activate the cyclin E-associated kinase, and this effect can be linked to an increased association of the cdk inhibitors, p21 and p27. These data describe a striking convergence in the cell cycle controls used by the two major signal transduction systems responsible for normal and abnormal cell growth. Taken together with our previous studies showing adhesion-dependent expression of cyclin A, they also establish the cell cycle basis for explaining the combined requirement for growth factors and the extracellular matrix in transit through the Rb checkpoint, entry into S phase, and anchorage-dependent growth.

The N-terminus of MDM2 proto-oncoprotein interacts with p53 and down modulates p53 activity by inhibiting transcriptional activity and promoting p53 degradation. MDMX is structurally related to MDM2 and also binds to p53. However, the function of MDMX has not been clarified yet. We found that MDM2 hetero-oligomerized with MDMX through their Cterminal RING finger domains. Yeast two-hybrid analysis revealed that the hetero-oligomerization between MDMX and MDM2 was more stable than the homo-oligomerization of each protein. MDM2 has been shown to be degraded by the ubiquitinproteasome pathway, while MDMX was a stable protein.Interaction of MDMX with MDM2 through the C-terminal RING finger domains resulted in inhibiting degradation of MDM2. These data indicate that MDMX functions as a regulator of MDM2.z 1999 Federation of European Biochemical Societies.

Cell adhesion has an essential role in regulating proliferation during the G1 phase of the cell cycle, and loss of this adhesion requirement is a classic feature of oncogenic transformation. The appearance of cyclin A messenger RNA and protein in late G1 was dependent on cell adhesion in both NRK and NIH 3T3 fibroblasts. In contrast, the expression of Cdc2, Cdk2, cyclin D1, and cyclin E was independent of adhesion in both cell lines. Transfection of NRK cells with a cyclin A complementary DNA resulted in adhesion-independent accumulation of cyclin A protein and cyclin A-associated kinase activity. These transfected cells also entered S phase and complete multiple rounds of cell division in the absence of cell adhesion. Thus, cyclin A is a target of the adhesion-dependent signals that control cell proliferation.

Abstract. The RCC1 gene, a regulator for the onset of chromosome condensation was found to encode a protein with a molecular mass of 45 kD, determined using the antibody against the synthetic peptides prepared according to the amino acid sequence of the putative RCC1 protein. The p45 located in the nuclei was released from the isolated nuclei, either by DNase I digestion or by treatment with 0.3 M NaCI. Consistently, p45 bound to the DNA-cellulose column was eluted with 0.3 M NaCI. After sequential treatment with DNase I and 2 M NaCI, almost all of the RCC1 protein were released from the nuclei. Thus, RCC1 protein locates on the chromatin and is not a component of the nuclear matrix. In mitotic cells, 1M5 is dispersed into the cytoplasm. Presumably, RCC1 protein plays a role in regulating the onset of chromosome condensation, at the level of transcription or of mRNA maturation.

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