A splice variant of Skp2 is retained in the cytoplasm and fails to direct cyclin D1 ubiquitination in the uterine cancer cell line SK-UT.

Cyclin D1 is an important regulator of the transition from G1 into S phase of the cell cycle. The level to which cyclin D1 accumulates is tightly regulated. One mechanism contributing to the control of cyclin D1 levels is the regulation of its ubiquitination. SK-UT-1B cells are deficient in the degradation of D-type cyclins. We show here that p27, a substrate of the SCF(Skp2) ubiquitin ligase complex, is coordinately stabilized in SK-UT-1B cells. Further, we show that expression of Skp2 in SK-UT-1B cells rescues the cyclin D1 and p27 degradation defect observed in this cell line. These results therefore indicate that the SCF(Skp2) ubiquitin ligase complex affects the ubiquitination of cyclin D1. In addition, we show that SK-UT-1B cells express a novel splice variant of Skp2 that localizes to the cytoplasm and that cyclin D1 ubiquitination takes place in the nucleus. We propose that the translocation of Skp2 into the nucleus is required for the ubiquitination of cyclin D1 and that the absence of the SCF(Skp2) complex in the nucleus of SK-UT-1B cells is the mechanism underlying the ubiquitination defect observed in this cell line. Finally, our data indicates that differential splicing of F-box proteins may represent an additional level of regulation of the F-box mediated ubiquitination pathway.

Role of MEKK2-MEK5 in the regulation of TNF-alpha gene expression and MEKK2-MKK7 in the activation of c-Jun N-terminal kinase in mast cells.

Cross-linking of the high-affinity IgE receptor (FcepsilonRI) on mast cells with IgE and multivalent antigen triggers mitogen-activated protein (MAP) kinase activation and cytokine gene expression. We report here that MAP kinase kinase 4 (MKK4) gene disruption does not affect either MAP kinase activation or cytokine gene expression in response to cross-linking of FcepsilonRI in embryonic stem cell-derived mast cells. MKK7 is activated in response to cross-linking of FcepsilonRI, and this activation is inhibited by MAP/ERK kinase (MEK) kinase 2 (MEKK2) gene disruption. In addition, expression of kinase-inactive MKK7 in the murine mast cell line MC/9 inhibits c-Jun NH(2)-terminal kinase (JNK) activation in response to cross-linking of FcepsilonRI, whereas expression of kinase-inactive MKK4 does not affect JNK activation by this stimulus. However, FcepsilonRI-induced activation of the tumor necrosis factor-alpha (TNF-alpha) gene promoter is not affected by expression of kinase-inactive MKK7. We describe an alternative pathway by which MEKK2 activates MEK5 and big MAP kinase1/extracellular signal-regulated kinase 5 in addition to MKK7 and JNK, and interruption of this pathway inhibits TNF-alpha promoter activation. These findings suggest that JNK activation by antigen cross-linking is dependent on the MEKK2-MKK7 pathway, and cytokine production in mast cells is regulated in part by the signaling complex MEKK2-MEK5-ERK5.

E2F4 is essential for normal erythrocyte maturation and neonatal viability.

The retinoblastoma protein (pRB) plays a key role in the control of normal development and proliferation through the regulation of the E2F transcription factors. We generated a mutant mouse model to assess the in vivo role of the predominant E2F family member, E2F4. Remarkably, loss of E2F4 had no detectable effect on either cell cycle arrest or proliferation. However, E2F4 was essential for normal development. E2f4-/- mice died of an increased susceptibility to opportunistic infections that appeared to result from craniofacial defects. They also displayed a variety of erythroid abnormalities that arose from a cell autonomous defect in late stage maturation. This suggests that E2F4 makes a major contribution to the control of erythrocyte development by the pRB tumor suppressor.

In vivo immunological function of mast cells derived from embryonic stem cells: an approach for the rapid analysis of even embryonic lethal mutations in adult mice in vivo.

An important goal of tissue engineering is to achieve reconstitution of specific functionally active cell types by transplantation of differentiated cell populations derived from normal or genetically altered embryonic stem cells in vitro. We find that mast cells derived in vitro from wild-type or genetically manipulated embryonic stem cells can survive and orchestrate immunologically specific IgE-dependent reactions after transplantation into mast cell-deficient Kit(W)/Kit(W-v) mice. These findings define a unique approach for analyzing the effects of mutations of any genes that are expressed in mast cells, including embryonic lethal mutations, in vitro or in vivo.

Protein-damaging stresses activate c-Jun N-terminal kinase via inhibition of its dephosphorylation: a novel pathway controlled by HSP72.

Various stresses activate the c-Jun N-terminal kinase (JNK), which is involved in the regulation of many aspects of cellular physiology, including apoptosis. Here we demonstrate that in contrast to UV irradiation, heat shock causes little or no stimulation of the JNK-activating kinase SEK1, while knocking out the SEK1 gene completely blocks heat-induced JNK activation. Therefore, we tested whether heat shock activates JNK via inhibition of JNK dephosphorylation. The rate of JNK dephosphorylation in unstimulated cells was high, and exposure to UV irradiation, osmotic shock, interleukin-1, or anisomycin did not affect this process. Conversely, exposure of cells to heat shock and other protein-damaging conditions, including ethanol, arsenite, and oxidative stress, strongly reduced the rate of JNK dephosphorylation. Under these conditions, we did not observe any effects on dephosphorylation of the homologous p38 kinase, suggesting that suppression of dephosphorylation is specific to JNK. Together, these data indicate that activation of JNK by protein-damaging treatments is mediated primarily by inhibition of a JNK phosphatase(s). Elevation of cellular levels of the major heat shock protein Hsp72 inhibited a repression of JNK dephosphorylation by these stressful treatments, which explains recent reports of the suppression of JNK activation by Hsp72.

SEK1 deficiency reveals mitogen-activated protein kinase cascade crossregulation and leads to abnormal hepatogenesis.

SEK1 (MKK4/JNKK) is a mitogen-activated protein kinase activator that has been shown to participate in vitro in two stress-activated cascades terminating with the SAPK and p38 kinases. To define the role of SEK1 in vivo, we studied stress-induced signaling in SEK1(-/-) embryonic stem and fibroblast cells and evaluated the phenotype of SEK1(-/-) mouse embryos during development. Studies of SEK1(-/-) embryonic stem cells demonstrated defects in stimulated SAPK phosphorylation but not in the phosphorylation of p38 kinase. In contrast, SEK1(-/-) fibroblasts exhibited defects in both SAPK and p38 phosphorylation, demonstrating that crosstalk exists between the stress-activated cascades. Tumor necrosis factor alpha and interleukin 1 stimulation of both stress-activated cascades are severely affected in the SEK1(-/-) fibroblast cells. SEK1 deficiency leads to embryonic lethality after embryonic day 12.5 and is associated with abnormal liver development. This phenotype is similar to c-jun null mouse embryos and suggests that SEK1 is required for phosphorylation and activation of c-jun during the organo-genesis of the liver.

SEK1/MKK4 is required for maintenance of a normal peripheral lymphoid compartment but not for lymphocyte development.

SAPK is a member of the group of evolutionary conserved stress-activated kinases that mediate control of cellular death and proliferation. In lymphocytes, the SAPK pathway has been implicated in signaling from antigen, costimulatory, and death receptors; SEK1, which directly activates SAPK, is required for early embryonic development and has also been reported to be essential for normal lymphocyte development. In contrast to the latter findings, we have used RAG-2-deficient blastocyst complementation to show that SEK1-deficient embryonic stem cells support unimpaired T and B lymphocyte development. Moreover, mature SEK1-deficient lymphocytes are capable of SAPK activation. Surprisingly, however, aging SEK1-deficient chimeric mice frequently develop lymphadenopathy and polyclonal B and T cell expansions. Thus, SEK1 is not required for lymphocyte development, but is required for maintaining peripheral lymphoid homeostasis.

An STS-based map of the human genome.

A physical map has been constructed of the human genome containing 15,086 sequence-tagged sites (STSs), with an average spacing of 199 kilobases. The project involved assembly of a radiation hybrid map of the human genome containing 6193 loci and incorporated a genetic linkage map of the human genome containing 5264 loci. This information was combined with the results of STS-content screening of 10,850 loci against a yeast artificial chromosome library to produce an integrated map, anchored by the radiation hybrid and genetic maps. The map provides radiation hybrid coverage of 99 percent and physical coverage of 94 percent of the human genome. The map also represents an early step in an international project to generate a transcript map of the human genome, with more than 3235 expressed sequences localized. The STSs in the map provide a scaffold for initiating large-scale sequencing of the human genome.

Kiz-1, a protein with LIM zinc finger and kinase domains, is expressed mainly in neurons.

The olfactory epithelium is the only neuronal tissue capable of generating new neurons during adult life and hence must express genes responsible for this phenomenon. Therefore, we have used mRNA from immortalized olfactory epithelial cells to search for novel protein tyrosine kinases by polymerase chain reaction, using as primers conserved sequences from the catalytic domain of known kinase genes. A full-length complementary DNA clone corresponding to one such polymerase chain reaction product was isolated and sequenced. This complementary DNA, designated Kiz-1, encodes a protein containing two prominent domains; the NH2-terminal region contains a cysteine/histidine-rich moiety previously identified as a zinc-finger domain in proteins of the LIM family, while the COOH-terminus contains a kinase domain. Kiz-1 is expressed mainly in the brain of adult mice but also in a range of cultured cell lines, regardless of their tissue of origin. Immunohistochemical studies on adult mouse brain demonstrated that Kiz-1 is expressed exclusively in neurons, not in astrocytes or oligodendrocytes. In the developing embryo, however, Kiz-1 is expressed in all tissues. In COS cells transfected with Kiz-1 complementary DNA and in the immortalized olfactory epithelial cells, Kiz-1 was found mainly in the cytoplasm, but in neurons of the adult brain, it resided also in the nucleus. Two Kiz-1 mRNA species are expressed in cell lines as well as in the murine and human brain. One transcript lacks a region of 60 nucleotides, which lies within the catalytic domain of the kinase and is encoded by a separate exon. Our results suggest that Kiz-1 may play distinct roles in dividing cells and in differentiated neurons.