Faculty Bibliography

Cyclin A/Cdk2 plays an important role during S and G2/M phases of the eukaryotic cell cycle, but the mechanisms by which it regulates cell cycle events are not fully understood. We have biochemically purified and identified SCAPER, a novel protein that specifically interacts with cyclin A/Cdk2 in vivo. Its expression is cell cycle independent, and it associates with cyclin A/Cdk2 at multiple phases of the cell cycle. SCAPER localizes primarily to the endoplasmic reticulum. Ectopic expression of SCAPER sequesters cyclin A from the nucleus and results specifically in an accumulation of cells in M phase of the cell cycle. RNAi-mediated depletion of SCAPER decreases the cytoplasmic pool of cyclin A and delays the G1/S phase transition upon cell cycle re-entry from quiescence. We propose that SCAPER represents a novel cyclin A/Cdk2 regulatory protein that transiently maintains this kinase in the cytoplasm. SCAPER could play a role in distinguishing S phase- from M phase-specific functions of cyclin A/Cdk2

The centrosome is an integral component of the eukaryotic cell cycle machinery, yet very few centrosomal proteins have been fully characterized to date. We have undertaken a series of biochemical and RNA interference (RNAi) studies to elucidate a role for CP110 in the centrosome cycle. Using a combination of yeast two-hybrid screens and biochemical analyses, we report that CP110 interacts with two different Ca(2+)-binding proteins, calmodulin (CaM) and centrin, in vivo. In vitro binding experiments reveal a direct, robust interaction between CP110 and CaM and the existence of multiple high-affinity CaM-binding domains in CP110. Native CP110 exists in large ( approximately 300 kDa to 3 MDa) complexes that contain both centrin and CaM. We investigated a role for CP110 in CaM-mediated events using RNAi and show that its depletion leads to a failure at a late stage of cytokinesis and the formation of binucleate cells, mirroring the defects resulting from ablation of either CaM or centrin function. Importantly, expression of a CP110 mutant unable to bind CaM also promotes cytokinesis failure and binucleate cell formation. Taken together, our data demonstrate a functional role for CaM binding to CP110 and suggest that CP110 cooperates with CaM and centrin to regulate progression through cytokinesis

Since their initial discovery in yeast, cyclin-dependent kinases have proven to be universal regulators of the cell cycle in all eukaryotes. In unicellular eukaryotes, cell cycle progression is principally governed by one catalytic subunit (cyclin-dependent kinase) that pairs with cell cycle-specific regulatory subunits known as cyclins. Progression through a specific phase of the cell cycle is under the control of a specific class of cyclin. Cell cycle control in multicellular eukaryotes has an additional layer of complexity, as multiple CDKs and cyclins are required. In this review, we will discuss recent advances in the area of cyclins and CDKs, with emphasis on the role of the mammalian proteins in cell cycle control at the cellular and at the organismal level. Many recent surprises have come to light recently as a result of genetic manipulation of cells and mice, and these findings suggest that our understanding of the intricacies of the cell cycle is still rudimentary at best

Mitogen-activated protein kinases/extracellular signal regulated kinases (MAPKs/ERKs) are typically thought to be soluble cytoplasmic enzymes that translocate to the nucleus subsequent to their phosphorylation by their activating kinases or mitogen-activated protein/extracellular signal regulated kinase kinase. We report here the first example of nuclear translocation of a MAPK that occurs via temporally regulated exit from a membranous organelle. Confocal microscopy examining the subcellular localization of ERK3 in several cell lines indicated that this enzyme was targeted to the Golgi/endoplasmic reticulum Golgi intermediate compartment. Deletion analysis of green fluorescent protein (GFP)-ERK3 uncovered a nuclear form that was carboxy-terminally truncated and established a Golgi targeting motif at the carboxy terminus. Immunoblot analysis of cells treated with the proteasome inhibitor MG132 further revealed two cleavage products, suggesting that in vivo, carboxy-terminal cleavage of the full-length protein controls its subcellular localization. In support of this hypothesis, we found that deletion of a small region rich in acidic residues within the carboxy terminus eliminated both the cleavage and nuclear translocation of GFP-ERK3. Finally, cell cycle synchronization studies revealed that the subcellular localization of ERK3 is temporally regulated. These data suggest a novel mechanism for the localization of an MAPK family member, ERK3, in which cell cycle-regulated, site-specific proteolysis generates the nuclear form of the protein

Palytoxin is a novel skin tumor promoter that does not activate protein kinase C. Previous studies demonstrated that palytoxin stimulates a sodium-dependent signaling pathway that activates the c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK) in Swiss 3T3 fibroblasts. In this study we show that a JNK kinase known as the stress-activated protein kinase/extracellular signal-regulated kinase-1 (SEK1) plays an important role in the regulation of JNK by palytoxin. We found that palytoxin stimulates the sustained activation of both JNK and SEK1 in COS7 and HeLa cells. Transiently expressed SEK1 isolated from palytoxin-treated cells can phosphorylate and activate JNK, which, in turn, can phosphorylate c-Jun. Furthermore, expression of a dominant negative mutant of SEK1 blocks activation of JNK by palytoxin. Sodium appears to play an important role in the regulation of JNK and SEK1 by palytoxin. Activation of JNK and SEK1 by palytoxin, but not anisomycin, requires extracellular sodium. Complementary studies showed that the sodium ionophore gramicidin can mimic palytoxin by regulating JNK and SEK1 through a sodium-dependent mechanism. Collectively, these results demonstrate that palytoxin stimulates a sodium-dependent signaling pathway that activates the SEK1/JNK/c-Jun protein kinase cascade

The pRB-related proteins p107 and p130 are thought to suppress growth in part through their associations with two important cell cycle kinases, cyclin A-cdk2 and cyclin E-cdk2, and transcription factor E2F. Although each protein plays a critical role in cell proliferation, the functional consequences of the association among growth suppressor, cyclin-dependent kinase, and transcription factor have remained elusive. In an attempt to understand the biochemical properties of such complexes, we reconstituted each of the p130-cyclin-cdk2 and p107-cyclin-cdk2 complexes found in vivo with purified, recombinant proteins. Strikingly, stoichiometric association of p107 or p130 with either cyclin E-cdk2 or cyclin A-cdk2 negated the activities of these kinases. The results of our experiments suggest that inhibition does not result from substrate competition or loss of cdk2 activation. Kinase inhibitory activity was dependent upon an amino-terminal region of p107 that is highly conserved with p130. Further, a role for this amino-terminal region in growth suppression was uncovered by using p107 mutants unable to bind E2F. To determine whether cellular complexes might display similar regulatory properties, we purified p130-cyclin A-cdk2 complexes from human cells and found that such complexes exist in two forms, one that contains E2F-4-DP-1 and one that lacks the heterodimer. These endogenous complexes behaved like the in vitro-reconstituted complexes, exhibiting low levels of associated kinase activity that could be significantly augmented by dissociation of p130. The results of these experiments suggest a mechanism whereby p130 and p107 suppress growth by inhibiting important cell cycle kinases

Ligation of the cell surface receptor Fas/APO-1 (CD95) by its specific ligand or by anti-Fas antibodies rapidly induces apoptosis in susceptible cells. To characterize the molecular events involved in Fas-induced apoptosis, we examined the contribution of two subgroups of the mitogen-activated protein (MAP) kinase family, the Jun kinases or stress-activated protein kinases (JNKs/SAPKs) and the extracellular signal-regulated kinases (ERKs), in a Fas-sensitive neuroblastoma cell line. Here we show that both JNK and ERK protein kinases were activated upon Fas crosslinking through a Ras-dependent mechanism. Interference with either the JNK or ERK pathway by ectopic expression of dominant-interfering mutant proteins blocked Fas-mediated apoptosis. ERK activation was transient and associated with induced expression of the Fas receptor. In contrast, JNK activation was sustained and correlated with the onset of apoptosis. These data indicate that the ERK and the JNK groups of MAP kinases cooperate in the induction of cell death by Fas. Inhibition of Fas killing by an interleukin 1beta-converting enzyme (ICE)-like protease inhibitor peptide did not modify Fas-induced JNK activation upon Fas ligation. In contrast, changes in Bcl-2 level due to expression of sense and antisense vectors influenced the sensitivity to Fas killing and Fas-induced JNK activation

Although a significant amount of evidence has demonstrated that there are intimate connections between transcriptional controls and cell cycle regulation, the precise mechanisms underlying these connections remain largely obscure. A number of recent advances have helped to define how critical cell cycle regulators, such as the retinoblastoma family of tumor suppressor proteins and the cyclin-dependent kinases, might function on a biochemical level and how such mechanisms of action have been conserved not only in the regulation of transcription by all three RNA polymerases but also across species lines. In addition, the use of in vivo techniques has begun to explain how the activity of the E2F transcription factor family is tied to the cell cycle dependent expression of target genes

Eukaryotic cells respond to different extracellular stimuli by recruiting homologous signalling pathways that use members of the MEKK, MEK and ERK families of protein kinases. The MEKK-->MEK-->ERK core pathways of Saccharomyces cerevisiae may themselves be regulated by members of the STE20 family of protein kinases. Here we report specific activation of the mammalian stress-activated protein kinase (SAPK) pathway by germinal centre kinase (GCK), a human STE20 homologue. SAPKs, members of the ERK family, are activated in situ by inflammatory stimuli, including tumour-necrosis factor (TNF) and interleukin-1, and phosphorylate and probably stimulate the transactivation function of c-Jun. Although GCK is found in many tissues, its expression in lymphoid follicles is restricted to the cells of the germinal centre, where it may participate in B-cell differentiation. Activation of the SAPK pathway by GCK illustrates further the striking conservation of eukaryotic signalling mechanisms and defines the first physiological function of a mammalian Ste20

The stress-activated protein kinases (SAPKs), which are distantly related to the MAP kinases, are the dominant c-Jun amino-terminal protein kinases activated in response to a variety of cellular stresses, including treatment with tumour-necrosis factor-alpha and interleukin-beta (refs 1, 2). SAPK phosphorylation of c-Jun probably activates the c-Jun transactivation function. SAPKs are part of a signal transduction cascade related to, but distinct from, the MAPK pathway. We have now identified a novel protein kinase, called SAPK/ERK kinase-1 (SEK1), which is structurally related to the MAP kinase kinases (MEKs). SEK1 is a potent activator of the SAPKs in vitro and in vivo. An inactive SEK1 mutant blocks SAPK activation by extracellular stimuli without interfering with the MAPK pathway. Although alternative mechanisms of SAPK activation may exist, as an immediate upstream activator of the SAPKs, SEK1 further defines a signalling cascade that couples cellular stress agonists to the c-Jun transcription factor