Nobumoto Watanabe

Dependence of cyclin E-CDK2 kinase activity on cell anchorage.

Most nonmalignant cells are anchorage-dependent; they require substrate attachment for growth and, in some instances, survival. This requirement is lost on oncogenic transformation. The cyclin E-CDK2 complex, which is required for the G1-S transition of the cell cycle, was activated in late G1 phase in attached human fibroblasts, but not in fibroblasts maintained in suspension. In transformed fibroblasts the complex was active regardless of attachment. The lack of cyclin E-CDK2 activity in suspended cells appeared to result from increased expression of CDK2 inhibitors and a concomitant decrease in phosphorylation of CDK2 on threonine-160. Suppression of cyclin E-CDK2 activity may thus underlie the anchorage dependence of cell growth.

The 'second-codon rule' and autophosphorylation govern the stability and activity of Mos during the meiotic cell cycle in Xenopus oocytes.

The c‐mos proto‐oncogene product, Mos, functions in both early (germinal vesicle breakdown) and late (metaphase II arrest) steps during meiotic maturation in Xenopus oocytes. In the early step, Mos is only partially phosphorylated and metabolically unstable, while in the late step it is fully phosphorylated and highly stable. Using a number of Mos mutants expressed in oocytes, we show here that the instability of Mos in the early step is determined primarily by its penultimate N‐terminal residue, or by a rule referred to here as the ‘second‐codon rule’. We demonstrate that unstable Mos is degraded by the ubiquitin‐dependent pathway. In the late step, on the other hand, Mos is stabilized by autophosphorylation at Ser3, which probably acts to prevent the N‐terminus of Mos from being recognized by a ubiquitin‐protein ligase. Moreover, we show that Ser3 phosphorylation is essential for Mos to exert its full cytostatic factor (CSF) activity in fully mature oocytes. Thus, a few N‐terminal amino acids are primary determinants of both the metabolic stability and physiological activity of Mos during the meiotic cell cycle.

Env-derived gp55 gene of Friend spleen focus-forming virus specifically induces neoplastic proliferation of erythroid progenitor cells.

A group of retroviruses carrying truncated viral genes has recently been suggested as the cause of new patterns of diseases. One such virus is the replication defective component of the Friend murine leukemia virus (F‐MuLV) complex, called Friend spleen focus forming virus (F‐SFFV). This virus induces erythroblastosis, and a virion envelope‐related glycoprotein, gp55, encoded by F‐SFFV has been suggested as the pathogenic gene. The role of the gp55 gene is, however, yet unclear in the apparently multistep erythroleukemogenesis. By separately producing transgenic mice harboring the whole F‐SFFV DNA, the gp55 gene alone under the control of the retroviral long terminal repeat (LTR) and the gp55 gene under the control of cytoplasmic beta actin transcriptional regulatory unit, we show here that the gp55 gene is capable of inducing neoplastic proliferation of erythroid progenitor cells specifically in the absence of helper virus and other F‐SFFV sequences. Under the control of the viral LTR the gp55 expression was detected only in leukemic tissues, but under the control of cytoplasmic beta‐actin regulatory sequences, the gp55 was also expressed in a variety of normal tissues including preleukemic normal spleens. The development of erythroleukemia was suppressed under the genetic background of C57B1/6 mouse (resistant to F‐MuLV; Fv‐2rr), and required additional events even under the background of DDD mouse (susceptible to F‐MuLV; Fv‐2ss). The p53 and Spi‐1 genes were frequently aberrant in transplanted tumors and cell lines derived from them, but were not in primary leukemic spleens.

SCFβTrCP mediates stress-activated MAPK-induced Cdc25B degradation

Cdc25A, which is one of the three mammalian CDK-activating Cdc25 protein phosphatases (Cdc25A, B and C), is degraded through SCFβTrCP-mediated ubiquitylation following genomic insult; however, the regulation of the stability of the other two Cdc25 proteins is not well understood. Previously, we showed that Cdc25B is primarily degraded by cellular stresses that activate stress-activated MAPKs, such as Jun NH2-terminal kinase (JNK) and p38. Here, we report that Cdc25B was ubiquitylated by SCFβTrCP E3 ligase upon phosphorylation at two Ser residues in the βTrCP-binding-motif-like sequence D94AGLCMDSPSP104. Point mutation of these Ser residues to alanine (Ala) abolished the JNK-induced ubiquitylation by SCFβTrCP, and point mutation of DAG to AAG or DAA eradicated both βTrCP binding and ubiquitylation. Further analysis of the mode of βTrCP binding to this region revealed that the PEST-like sequence from E82SS to D94AG is crucially involved in both the βTrCP binding and ubiquitylation of Cdc25B. Furthermore, the phospho-mimetic replacement of all 10 Ser residues in the E82SS to SPSP104 region with Asp resulted in βTrCP binding. Collectively, these results indicate that stress-induced Cdc25B ubiquitylation by SCFβTrCP requires the phosphorylation of S101PS103P in the βTrCP-binding-motif-like and adjacent PEST-like sequences.

Correlation between Physiological and Transforming Activities of the c–mos Proto–oncogene Product and Identification of an Essential Mos Domain for These Activities

Using Xenopus eggs and NIH3T3 cells as assay systems, we have compared the physiological (i.e. maturation–inducing and cleavage–arresting) and in vitro transforming activities of the c–mos genes from various species as well as their mutant genes. These analyses show that the three biological activities all depend upon the intrinsic protein kinase activity of Mos and correlate well with each other. Furthermore, our results demonstrate that a well conserved N–terminal 14–amino acid sequence of Mos, termed the Mos–hox, is essential for all three activities. These results indicate that the in vitro transforming activity of Mos can he ascribed to the same kinase activity of Mos that exerts the physiological activities.