Kazuo Todokoro

PKA and MPF-Activated Polo-like Kinase Regulate Anaphase-Promoting Complex Activity and Mitosis Progression

Ubiquitin-mediated proteolysis is the key to cell cycle control. Anaphase-promoting complex/cyclosome (APC) is a ubiquitin ligase that targets cyclin B and factors regulating sister chromatid separation for proteolysis by the proteasome and, consequently, regulates metaphase-anaphase transition and exit from mitosis. Here we report that Cdc2-cyclin B-activated Polo-like kinase (Plk) specifically phosphorylates at least three components of APC and activates APC to ubiquitinate cyclin B in the in vitro-reconstituted system. Conversely, protein kinase A (PKA) phosphorylates two subunits of APC but suppresses APC activity. PKA is superior to Plk in its regulation of APC, and Plk activity peaks whereas PKA activity is falling at metaphase. These results indicate that Plk and PKA regulate mitosis progression by controlling APC activity.

Essential roles of KIF4 and its binding partner PRC1 in organized central spindle midzone formation

A number of proteins accumulate in the anaphase spindle midzone, but the interaction and precise role of these proteins in midzone organization remain obscure. Here, we found that the microtubule‐bundling protein PRC1 bound separately to the three motor proteins, KIF4, MKLP1 and CENP‐E, but not to the chromosomal passenger proteins. In KIF4‐deficient cells, the central spindle was disorganized, and all midzone‐associated proteins including PRC1 failed to concentrate at the midline, instead being dispersed along the loosened microtubule bundles of the central spindle. This suggests that KIF4 is essential for the organization of central spindles and for midzone formation. In PRC1‐deficient cells, no midzone was formed, KIF4 and CENP‐E did not localize to the disconnected half‐spindle, and MKLP1 and chromosomal passenger proteins localized to discrete subdomains near microtubule plus ends in the half‐spindle. Thus, PRC1 is required for interaction of the two half‐spindles and for localization of KIF4 and CENP‐E. These results suggest that KIF4 and its binding partner PRC1 play essential roles in the organization of central spindles and midzone formation.

Ste20-like kinase (SLK), a regulatory kinase for polo-like kinase (Plk) during the G2/M transition in somatic cells.

Activation of the cyclin‐dependent kinase cdc2‐cyclin B1 at the G2/M transition of the cell cycle requires dephosphorylation of threonine‐14 and tyrosine‐15 in cdc2, which in higher eukaryotes is brought about by the Cdc25C phosphatase. In Xenopus, there is evidence that a kinase cascade comprised of xPlkk1 and Plx1, the Xenopus polo‐like kinase 1, plays a key role in the activation of Cdc25C during oocyte maturation. In the mammalian somatic cell cycle, a polo‐like kinase homologue (Plk1) also functions during mitosis, but a kinase upstream of Plk is still unknown.

Thrombopoietin induces activation of at least two distinct signaling pathways

Thrombopoietin (Tpo) is a cytokine regulating megakaryocyte maturation and platelet formation. We studied Tpo‐induced signal transduction, and found that Tpo induces phosphorylation of adapter molecules Shc and Vav, and of serine/threonine kinases Raf‐1 and mitogen‐activated protein (MAP) kinases. Further, Tpo induced activation of Ras, MAP kinase kinase, MAP kinase and Pim‐1. Taken together with other observations, we concluded that Tpo induces the activation of at least two distinct signaling pathways, a specific Tyk2‐JAK2/STAT1‐STAT3‐STAT5 signaling cascade and a common Shc/Vav/Ras/Raf‐1/MAP kinase kinase/MAP kinase signaling cascade.

Targeted disruption of MAIL, a nuclear IκB protein, leads to severe atopic dermatitis-like disease

MAIL (molecule-possessing ankyrin repeats induced by lipopolysaccharide) is a nuclear IκB protein that is also termed interleukin-1-inducible nuclear ankyrin repeat protein or inhibitor of nuclear factor κB (IκB) ζ. In this study, we generated Mail–/– mice to investigate the roles of MAIL in whole organisms. Mail–/– mice grew normally until 4–8 weeks after birth, when they began to develop lesions in the skin of the periocular region, face, and neck. MAIL mRNA and protein were constitutively expressed in the skin of wild type controls, especially in the keratinocytes. Serum IgE was higher in Mail–/– mice than in normal. Histopathological analysis indicated that the Mail–/– skin lesions appeared to be atopic dermatitis (AD) eczema with inflammatory cell infiltration. In addition, markedly elevated expression of some chemokines such as thymus and activation-regulated chemokine was detected in the Mail–/– skin lesions, similar to that observed in the skin of patients with AD. In Mail–/– mice, MAIL-deficient keratinocytes might be activated to produce chemokines and induce intraepidermal filtration of inflammatory cells, resulting in the onset of the AD-like disease. These findings suggest that MAIL is an essential molecule for homeostatic regulation of skin immunity. The Mail–/– mouse is a valuable new animal model for research on AD.

Tec is involved in G protein-coupled receptor- and integrin-mediated signalings in human blood platelets

Tec is a non-receptor type tyrosine kinase which is tyrosine phosphorylated and activated upon stimulation of hematopoietic cells with various cytokines. The role of Tec in G protein-coupled receptor- and integrin-mediated signalings has not been elucidated. We therefore investigated the regulation of Tec in human blood platelets. Tec was rapidly tyrosine phosphorylated in response to platelet agonists which activate G protein-coupled receptors such as thromboxane A2 analog (U46619), thrombin, and thrombin receptor activating peptide (TRAP). TRAP-induced phosphorylation in Tec was significantly reduced under the conditions which abrogate fibrinogen binding to GP IIb–IIIa and subsequent platelet aggregation. However, TRAP induced significant levels of the phosphorylation even under these conditions and also in thrombasthenic platelets which lack functional GP IIb–IIIa molecules, suggesting that activation of G-protein-coupled receptor causes the phosphorylation. To clarify whether integrin engagement by itself causes the phosphorylation in Tec, we examined the state of the phosphorylation in platelets activated by integrin engagement. Platelet adhesion to immobilized fibrinogen or collagen induced significant levels of the phosphorylation. Furthermore, Tec was translocated to cytoskeleton in response to TRAP in a manner dependent on platelet aggregation, suggesting that Tec can be a component of integrin-mediated signalings. These results collectively indicate that Tec is involved in G protein-coupled receptor- and integrin-mediated signalings in human blood platelets.

Constitutive expression of exogenous c-myb gene causes maturation block in monocyte-macrophage differentiation

A nuclear protooncogene c-myb has been hypothesized to play an important role in hematopoiesis, but little is known about the physiological function of the c-myb gene products. To study the role of c-myb gene expression in monocyte-macrophage differentiation and proliferation, we introduced exogenous c-myb gene into murine myelomonocytic leukemia WEHI-3B(D+) cells which can be induced to differentiate into mature monocytes with granulocyte-colony stimulation factor (G-CSF) and actinomycin D. Expression of the transfected gene was found to result in elevated levels of c-myb transcripts, which were not subject to normal down-regulation by differentiation induction. This constitutive expression of c-myb gene allowed the c-myb transfectants to differentiate into promonocytes with G-CSF and actinomycin D, but blocked further maturation from promonocytes to mature monocytes. It is concluded that normal down-regulation of c-myb gene expression during monocyte-macrophage differentiation is required for the maturation of promonocytes to mature monocytes.

Tryptophan residue of Trp-Ser-X-Trp-Ser motif in extracellular domains of erythropoietin receptor is essential for signal transduction

The Trp-Ser-X-Trp-Ser motif commonly exists just outside the transmembrane domains of all cytokine receptors so far isolated. The role of this conserved motif in erythropoietin receptor was examined by assessing a series of mutant receptors on erythropoietin-induced signal transduction. Replacement of one of the two conserved Trp residues in the motif to Gly was found to completely abolish the binding of erythropoietin to the receptor and also to lose the ability to transduce the factor-dependent growth signal. While the mutants with one Ser residue converted to Gly or Ala retained full biological activities, the replacement of both conserved Ser residues diminished the functions of the receptor. Furthermore, the receptors lacking a part or all of the Trp-Ser-X-Trp-Ser motif did not respond to erythropoietin. The Trp-Ser-X-Trp-Ser motif, especially Trp residue, located in extracellular domains of the erythropoietin receptor thus appears to play a critical role in receptor-mediated signal transduction.

Sequential expression of proto-oncogenes during a mouse erythroleukemia cell differentiation

To investigate the possible involvement of proto-oncogenes in the regulation of cell differentiation and proliferation, their transcriptional levels were measured by Northern blot analysis during the process of dimethyl sulfoxide-induced differentiation of mouse erythroleukemia cells. The differentiation into erythrocytes was accompanied by sequential transient expression of the proto-oncogenes in the order of c-fos, c-myb, c-myc and c-k-ras, during the first 48 hr of differentiation induction. Following the transient expression of these proto-oncogenes, adult type sss-globin and embryonic type yl-globin gene transcripts appeared and accumulated in terminally differentiated cells. The expression of ten other proto-oncogenes examined here were not detected, nor dramatically changed during differentiation process.

Identification of human APC10/Doc1 as a subunit of anaphase promoting complex.

Anaphase-promoting complex or cyclosome (APC) is a ubiquitin ligase which specifically targets mitotic regulatory factors such as Pds1/Cut2 and cyclin B. Identification of the subunits of multiprotein complex APC in several species revealed the highly conserved composition of APC from yeast to human. It has been reported, however, that vertebrate APC is composed of at least eight subunits, APC1 to APC8, while budding yeast APC is constituted of at least 12 components, Apc1 to Apc13. It has not yet been clearly understood whether additional components found in budding yeast, Apc9 to Apc13, are actually composed of mammalian APC. Here we isolated and characterized human APC10/Doc1, and found that APC10/Doc1 binds to APC core subunits throughout the cell cycle. Further, it was found that APC10/Doc1 is localized in centrosomes and mitotic spindles throughout mitosis, while it is also localized in kinetochores from prophase to anaphase and in midbody in telophase and cytokinesis. These results strongly support the notion that human APC10/Doc1 may be one of the APC core subunits rather than the transiently associated regulatory factor.