Cdc2-related Kinase Research Articles

Preliminary Evidence that Conjugated Linoleic Acid Consumption Influences Global Gene Expression in the Lactating Human Mammary Gland

Conjugated linoleic acids (CLA) are octadecadienoic acid (18:2) isomers with conjugated double bonds. We are interested in understanding how CLA modifies gene expression in the human lactating mammary gland in terms of lipid synthesis, neoplasia, and host-pathogen interactions. As such, we have conducted a cross-over study in which lactating women (n = 3) consumed CLA (39.2% c9,t11-18:2; 38.5% t10,c12-18:2) and placebo (safflower oil) supplements (3 g) for 5 d. Exfoliated mammary epithelial cells were harvested from hind milk using immunomagnetic separation. Total RNA was extracted, amplified, labeled, and hybridized to Affymetrix Human Genome U133 Plus 2.0 GeneChips. Normalized, log-transformed data were analyzed using GeneSifter. Analysis found 129 transcripts affected by CLA; 64 and 65 down- and up-regulated, respectively. For example, CLA influenced (P < 0.05) expression of the following transcripts (placebo vs. CLA): MAPK kinase 6 (−0.28 ± 0.15 vs. −1.22 ± 0.21), TRAF4 (1.77 ± 0.15 vs. 1.16 ± 0.14), protein tyrosine phosphatase (0.38 ± 0.01 vs. 1.03 ± 0.14), and CDC2-related protein kinase 7 (1.35 ± 0.15 vs. 2.01 ± 0.15). Our data suggest that CLA influences cellular and molecular processes including cell cycle regulation, lipid metabolism, phosphorylation, transcription/translation, ion binding, ubiquitization, immune modulation, and intracellular trafficking in the mammary gland. Supported by the WSU Cancer Prevention and Research Center and NIH P20 RR15587

Trypanosoma cruzi Tcp12 CKS1 interacts with parasite CRKs and rescues the p13 SUC1 fission yeast mutant

The complex mechanism of cell division in trypanosomatids is not completely fully understood. CRKs (cdc2-related kinases), Cyclins and CKSs (cdc2-kinase subunit) are involved in the progression through the cell cycle. The CKS proteins were first described as components of the cell cycle machinery in yeast and their action has been implicated in the regulation of CDK function. In the present work we identified Tcp12 CKS1 a member of the CKS family in the parasite Trypanosoma cruzi. TcCKS1 is expressed in the three forms of T. cruzi. By using anti-Tcp12 CKS1 antiserum, protein kinase (PK) activities were immunoprecipitated. The PK activity level varies depending on the stage analyzed, being lower in trypomastigotes and thus suggesting that different stages have different CKS–CRK complexes. Moreover, these PK activities were inhibited by using Flavopiridol, a known CDKs inhibitor. Western blot analyses demonstrated that in the epimastigote stage, p12 CKS1 stably interacts with TcCRK1 and TcCRK3. In addition, Tcp12 CKS1 was able to rescue the p13 SUC1 null mutant of S. pombe. The functional complementation between the CKS proteins of two evolutionary distant organisms supports the role of Tcp12 CKS1 as a key regulator in T. cruzi cell cycle.

Pbcrk-1, the Plasmodium berghei orthologue of P. falciparum cdc-2 related kinase-1 (Pfcrk-1), is essential for completion of the intraerythrocytic asexual cycle

The molecular mechanisms underlying gametocytogenesis in malaria parasites are not understood. Plasmodium falciparum cdc2-related kinase 1 ( pfcrk- 1), a gene that is expressed predominantly in gametocytes, bears homology to the PITSLRE subfamily of cyclin-dependent kinases and has been hypothesized to function as a negative regulator of the cell cycle. We attempted to knock-out pbcrk-1, the P. berghei orthologue of pfcrk- 1, but were unable to recover P. berghei parasites with a disrupted pbcrk-1 locus. In contrast, an integration event at this locus that did not result in a loss-of-function of the pbcrk-1 gene was readily observed. This strongly suggests that a functional pbcrk- 1 gene product is essential to intraerythrocytic asexual multiplication.

Beyond the cell cycle: A new role for Cdk6 in differentiation

Over 10 years ago, cdk6 was identified as a new member in a family of vertebrate cdc-2 related kinases. This novel kinase was found to partner with the D-type cyclins and to possess pRb kinase activity in vitro and has since been understood to function solely as a pRb kinase in the regulation of the G(1) phase of the cell cycle. In the past 2 years, several independent studies in multiple cell types have indicated a novel role for cdk6 in differentiation. For example, cdk6 expression must be reduced to allow proper osteoblast and osteoclast differentiation, forced cdk6 expression blocked differentiation of mouse erythroid leukemia cells and cdk6 expression in primary astrocytes favors the expression of progenitor cell markers. Since exit from the cell cycle is a necessary step in terminal differentiation, down-regulation of a mitogenic factor may be expected in this process, however it is surprising that this association has not been previously uncovered and that it is apparently not shared with cdk4, long understood to be a functional homolog of cdk6. The mechanism of cdk6 function in differentiation is not understood, but it may extend beyond the established role of cdk6 as a pRb kinase. As this story unfolds it will be important to discover if the function of cdk6 in differentiation is pRb-dependent or pRb-independent, since pRb has long been established as a key factor in initiating and maintaining cell cycle exit during differentiation.

Distinct cytoskeletal modulation and regulation of G1-S transition in the two life stages ofTrypanosoma brucei

Procyclic-form Trypanosoma brucei is arrested in G1 phase with extended and/or branched posterior morphology when expression of its cdc2-related kinases 1 and 2 (CRK1 and CRK2) is knocked down by RNA interference. Transmission electron microscopy indicated that the mitochondrion in the cell is also extended and branched and associated with cortical microtubules in each elongated/branched posterior end. This posterior extension is apparently driven by the growing microtubule corset, as it can be blocked by rhizoxin, an inhibitor of microtubule assembly. In the bloodstream form of T. brucei, however, a knockdown of CRK1 and CRK2 resulted only in an enrichment of cells in G1 phase without cessation of DNA synthesis or elongated/branched posterior ends. A triple knockdown of CRK1, CRK2 and CycE1/CYC2 in the bloodstream form resulted in 15% of the cells arrested in G1 phase, but no cells had an abnormal posterior morphology. The double and triple knockdown bloodstream-form cells were differentiated in vitro into the procyclic form, and the latter thus generated bore the typical morphology of a procyclic form without an extended/branched posterior end, albeit arrested in the G1 phase as the bloodstream-form precursor. There is thus a major distinction in the mechanisms regulating G1-S transition and posterior morphogenesis between the two life stages of T. brucei.

Activation of a Nuclear Cdc2-Related Kinase within a Mitogen-Activated Protein Kinase-Like TDY Motif by Autophosphorylation and Cyclin-Dependent Protein Kinase-Activating Kinase

Male germ cell-associated kinase (MAK) and intestinal cell kinase (ICK) are nuclear Cdc2-related kinases with nearly identical N-terminal catalytic domains and more divergent C-terminal noncatalytic domains. The catalytic domain is also related to mitogen-activated protein kinases (MAPKs) and contains a corresponding TDY motif. Nuclear localization of ICK requires subdomain XI and interactions of the conserved Arg-272, but not kinase activity or, surprisingly, any of the noncatalytic domain. Further, nuclear localization of ICK is required for its activation. ICK is activated by dual phosphorylation of the TDY motif. Phosphorylation of Tyr-159 in the TDY motif requires ICK autokinase activity but confers only basal kinase activity. Full activation requires additional phosphorylation of Thr-157 in the TDY motif. Coexpression of ICK with constitutively active MEK1 or MEK5 fails to increase ICK phosphorylation or activity, suggesting that MEKs are not involved. ICK and MAK are related to Ime2p in budding yeast, and cyclin-dependent protein kinase-activating kinase Cak1p has been placed genetically upstream of Ime2p. Recombinant Cak1p phosphorylates Thr-157 in the TDY motif of recombinant ICK and activates its activity in vitro. Coexpression of ICK with wild-type CAK1 but not kinase-inactive CAK1 in cells also increases ICK phosphorylation and activity. Our studies establish ICK as the prototype for a new group of MAPK-like kinases requiring dual phosphorylation at TDY motifs.

Pairwise Knockdowns of cdc2-Related Kinases (CRKs) in Trypanosoma brucei Identified the CRKs for G 1 /S and G 2 /M Transitions and Demonstrated Distinctive Cytokinetic Regulations between Two Developmental Stages of the Organism

Expression of the cdc2-related kinase 3 (CRK3) together with expression of CRK1, -2, -4, or -6, were knocked down in pairs in the procyclic and bloodstream forms of Trypanosoma brucei, using the RNA interference technique. Double knockdowns of CRK3 and CRK2, CRK4, or CRK6 exerted significant growth inhibition and enriched the cells in G2/M phase, whereas a CRK3 plus CRK1 (CRK3 + CRK1) knockdown arrested cells in both G1/S and G2/M transitions. Thus, CRK1 and CRK3 are apparently the kinases regulating the G1/S and G2/M checkpoint passages, respectively, whereas the other CRKs are probably playing only minor roles in cell cycle regulation. A CRK1 + CRK2 knockdown in the procyclic form was found to cause aberrant posterior cytoskeletal morphogenesis (X. M. Tu and C. C. Wang, Mol. Biol. Cell 16:97-105, 2005). A CRK3 + CRK2 knockdown, however, did not lead to such a change, suggesting that CRK2 depletion can lead to the abnormal morphogenesis only when procyclic-form cells are arrested in the G1 phase. The G2/M-arrested procyclic form produces up to 20% stumpy anucleated cells (zoids) in the population, suggesting that cytokinesis and cell division are not blocked by mitotic arrest but are apparently driven to completion by the kinetoplast cycle. In the bloodstream form, however, G2/M arrest resulted in little zoid formation but, instead, enriched a population of cells each containing multiple kinetoplasts, basal bodies, and flagella and an aggregate of multiple nuclei, indicating failure in entering cytokinesis. The two different cytokinetic regulations between two distinct stage-specific forms of the same organism may provide an interesting and useful model for further understanding the evolution of cytokinetic control among eukaryotes.

Protein expression pattern of CDK11p58 during testicular development in the mouse

Protein kinases are important signalling molecules critical for normal cell growth and development. CDK11(p58) is a p34(cdc2) related protein kinase, and plays an important role in normal cell cycle progression. In this study, we mainly characterized the protein expression of CDK11(p58) during postnatal development in mouse testes and examined the cellular localization of CDK11(p58) and cyclinD3, which was associated with CDK11(p58) in mammalian cells. Western blot analysis revealed that CDK11(p58) was present in the early stages of development. It gradually increased and reached a peak in adult testes. The protein expression of CDK11(p58) was further analysed by immunohistochemistry due to its developmentally regulated expression. The variable immunostaining patterns of CDK11(p58) were visualized during different developmental periods and, in adult mouse, different stages of seminiferous tubules. CDK11(p58) expression was detected in proliferating germ cells in the early stages of developing testes. In adult testes, the protein was expressed in pachytene primary spermatocytes from stage VII to XI of spermatogenesis and in postmeiotic spermatids in all stages at different levels. The colocalization of CDK11(p58) and cyclinD3 in the adult testis was revealed by immunofluorescence analysis.

Downregulation of β1,4-galactosyltransferase 1 inhibits CDK11 p58-mediated apoptosis induced by cycloheximide

Cyclin-dependent kinase 11 (CDK11; also named PITSLRE) is part of the large family of p34 cdc2-related kinases whose functions appear to be linked with cell cycle progression, tumorigenesis, and apoptotic signaling. The mechanism that CDK11 p58 induces apoptosis is not clear. Some evidences suggested β1,4-galactosyltransferase 1 (β1,4-GT 1) might participate in apoptosis induced by CDK11 p58. In this study, we demonstrated that ectopically expressed β1,4-GT 1 increased CDK11 p58-mediated apoptosis induced by cycloheximide (CHX). In contrast, RNAi-mediated knockdown of β1,4-GT 1 effectively inhibited apoptosis induced by CHX in CDK11 p58-overexpressing cells. For example, the cell morphological and nuclear changes were reduced; the loss of cell viability was prevented and the number of cells in sub-G1 phase was decreased. Knock down of β1,4-GT 1 also inhibited the release of cytochrome c from mitochondria and caspase-3 processing. Therefore, the cleavage of CDK11 p58 by caspase-3 was reduced. We proposed that β1,4-GT 1 might contribute to the pro-apoptotic effect of CDK11 p58. This may represent a new mechanism of β1,4-GT 1 in CHX-induced apoptosis of CDK11 p58-overexpressing cells.

Coupling of Posterior Cytoskeletal Morphogenesis to the G1/S Transition in theTrypanosoma bruceiCell Cycle

The expression levels of four Cdc2-related kinases (CRK1, 2, 4, and 6) in the procyclic form of Trypanosoma brucei were knocked down in pairs using the RNA interference (RNAi) technique. A double knockdown of CRK1 and CRK2 resulted in arrested cell growth in the G1 phase accompanied by an apparent cessation of nuclear DNA synthesis. The arrested cells became elongated at the posterior end like the G1-phase cells generated by knockdown of CycE1/CYC2 in a previous study. However, approximately 5% of the G1 cells in the current study also possessed multiply branched posterior ends, which have not previously been observed in T. brucei. DAPI and immunofluorescence staining showed a single nucleus, kinetoplast, basal body, and flagellum in the anterior portion of each G1 cell. The split and grossly extended posterior ends were heavily stained with antibodies to tyrosinated alpha-tubulin, suggesting an accumulation of newly synthesized microtubules. A significant population of anucleate cells (zoids), apparently derived from kinetoplast-dictated cytokinesis and cell division of the G1 cells, also had extended and branched posterior ends filled with newly synthesized microtubules. This continued posterior extension of microtubules in the G1 cells and zoids suggests that CRK1 and CRK2 exert a coordinated control on G1/S passage and the limited growth of the microtubule corset toward the posterior end. This connection may provide a new insight into the mechanism of morphological maintenance of an ancient protist during its cell cycle progression.

NpkA, a cdc2-related kinase from Aspergillus nidulans, interacts with the UvsBATR kinase

NpkA, a cdc2-related kinase from Aspergillus nidulans, interacts with the UvsBATR kinase

The Aspergillus nidulans npkA gene encodes a Cdc2-related kinase that genetically interacts with the UvsBATR kinase.

The DNA damage response is a protective mechanism that ensures the maintenance of genomic integrity. We have used Aspergillus nidulans as a model system to characterize the DNA damage response caused by the antitopoisomerase I drug, camptothecin. We report the molecular characterization of a p34Cdc2-related gene, npkA, from A. nidulans. The npkA gene is transcriptionally induced by camptothecin and other DNA-damaging agents, and its induction in the presence of camptothecin is dependent on the uvsBATR gene. There were no growth defects, changes in developmental patterns, increased sensitivity to DNA-damaging agents, or effects on septation or growth rate in the A. nidulans npkA deletion strain. However, the DeltanpkA mutation can partially suppress HU sensitivity caused by the DeltauvsBATR and uvsD153ATRIP checkpoint mutations. We demonstrated that the A. nidulans uvsBATR gene is involved in DNA replication and the intra-S-phase checkpoints and that the DeltanpkA mutation can suppress its intra-S-phase checkpoint deficiency. There is a defect in both the intra-S-phase and DNA replication checkpoints due to the npkA inactivation when DNA replication is slowed at 6 mm HU. Our results suggest that the npkA gene plays a role in cell cycle progression during S-phase as well as in a DNA damage signal transduction pathway in A. nidulans.

The Trypanosoma brucei Cyclin, CYC2, Is Required for Cell Cycle Progression through G1 Phase and for Maintenance of Procyclic Form Cell Morphology

CYC2 is an essential PHO80-like cyclin that forms a complex with the cdc2-related kinase CRK3 in Trypanosoma brucei. In both procyclic and bloodstream form T. brucei, knock-down of CYC2 by RNA interference (RNAi) led to an accumulation of cells in G(1) phase. Additionally, in procyclic cells, but not in bloodstream form cells, CYC2 RNAi induced a specific cell elongation at the posterior end. The G(1) block, as well as the posterior end elongation in the procyclic form, was irreversible once established. Staining for tyrosinated alpha-tubulin and morphometric analyses showed that the posterior end elongation occurred through active microtubule extension, with no repositioning of the kinetoplast. Hence, these cells can be classified as exhibiting the "nozzle" phenotype as has been described for cells that ectopically express TbZFP2, a zinc finger protein that is involved in the differentiation of the bloodstream form to procyclic form. Within the tsetse fly, procyclic trypanosomes differentiate to elongated mesocyclic cells. However, although mesocyclic trypanosomes isolated from tsetse flies also show active microtubule extension at the posterior end, the kinetoplast is coincidentally repositioned such that it always lies approximately midway between the nucleus and posterior end of the cell. Thus, in the procyclic form CYC2 has dual functionality and is required for both cell cycle progression through G(1) and for the maintenance of correct cell morphology, whereas in the bloodstream form only a role for CYC2 in G(1) progression is evident.

The Involvement of Two cdc2-related Kinases (CRKs) in Trypanosoma brucei Cell Cycle Regulation and the Distinctive Stage-specific Phenotypes Caused by CRK3 Depletion

Cyclin-dependent protein kinases are among the key regulators of eukaryotic cell cycle progression. Potential functions of the five cdc2-related kinases (CRK) in Trypanosoma brucei were analyzed using the RNA interference (RNA(i)) technique. In both the procyclic and bloodstream forms of T. brucei, CRK1 is apparently involved in controlling the G(1)/S transition, whereas CRK3 plays an important role in catalyzing cells across the G(2)/M junction. A knockdown of CRK1 caused accumulation of cells in the G(1) phase without apparent phenotypic change, whereas depletion of CRK3 enriched cells of both forms in the G(2)/M phase. However, two distinctive phenotypes were observed between the CRK3-deficient procyclic and bloodstream forms. The procyclic form has a majority of the cells containing a single enlarged nucleus plus one kinetoplast. There is also an enhanced population of anucleated cells, each containing a single kinetoplast known as the zoids (0N1K). The CRK3-depleted bloodstream form has an increased number of one nucleus-two kinetoplast cells (1N2K) and a small population containing aggregated multiple nuclei and multiple kinetoplasts. Apparently, these two forms have different mechanisms in cell cycle regulation. Although the procyclic form can be driven into cytokinesis and cell division by kinetoplast segregation without a completed mitosis, the bloodstream form cannot enter cytokinesis under the same condition. Instead, it keeps going through another G(1) phase and enters a new S phase resulting in an aggregate of multiple nuclei and multiple kinetoplasts in an undivided cell. The different leakiness in cell cycle regulation between two stage-specific forms of an organism provides an interesting and useful model for further understanding the evolution of cell cycle control among the eukaryotes.

Candida albicans Cdc37 interacts with the Crk1 kinase and is required for Crk1 production

Crk1, a Cdc2-related protein kinase from the human pathogenic fungus Candida albicans, plays an important role in hyphal development and virulence. To address its regulatory mechanisms, we searched for Crk1 interacting proteins by two-hybrid screening. A CDC37 ortholog ( CaCDC37) was cloned from the screening with the Crk1 kinase domain as the bait. The CaCdc37 interacted preferentially with the kinase domain of Crk1 (Crk1N) as shown by two-hybrid and immunoprecipitation experiments. CaCDC37 could complement a cdc37 thermosensitive mutant ( cdc37-34) of Saccharomyces cerevisiae. Importantly, Crk1 protein was hardly detectable in the cdc37-34 mutant at restrictive temperature. However, upon expression of CaCdc37 in the cdc37 mutant, Crk1 protein was detected even at restrictive temperature. Our data suggested that CaCdc37 was required for the production of Crk1 kinase. Like Cdc37 proteins of S. cerevisiae and higher eukaryotes, CaCdc37 might function as a molecular chaperone that stabilized Crk1 and other protein kinases in C. albicans. In support of this, CaSTI1 was identified from a two-hybrid screen with the full-length Crk1 as the bait. CaSti1 showed two-hybrid interactions with both Crk1 and the CaCdc37.

NKIAMRE, a novel conserved CDC2-related kinase with features of both mitogen-activated protein kinases and cyclin-dependent kinases

We report the cloning of the NKIAMRE gene located on human chromosome 5q31.1. It encodes a novel 52 kDa Cdc2-related kinase with a 1.5 kb open reading frame. Like MAP kinases, NKIAMRE contains a Thr–X–Tyr (TXY) motif in the activation loop domain. Similar to cdks, NKIAMRE contains the putative negative regulatory Ser14 and Tyr15 residues and the cyclin-binding motif, NKIAMRE, from which it derives its name. Human NKIAMRE has significant amino acid identity to related kinases in rat, mouse, Caenorhabditis elegans, and Drosophila, and is widely expressed in human tissues and cell lines. Confocal microscopy demonstrates that NKIAMRE localizes to the cytoplasm. NKIAMRE is activated by treatment of cells with phorbol 12-myristate 13-acetate. Mutation of the ATP-binding Lys-33 to arginine and the Thr–Glu–Tyr motif to Ala–Glu–Phe abolished its ability to phosphorylate myelin basic protein. NKIAMRE is a member of a conserved family of kinases with homology to both MAP kinases and cyclin-dependent kinases.

CDK9 is constitutively expressed throughout the cell cycle, and its steady-state expression is independent of SKP2.

CDK9 is a CDC2-related kinase and the catalytic subunit of the positive-transcription elongation factor b and the Tat-activating kinase. It has recently been reported that CDK9 is a short-lived protein whose levels are regulated during the cell cycle by the SCF(SKP2) ubiquitin ligase complex (R. E. Kiernan et al., Mol. Cell. Biol. 21:7956-7970, 2001). The results presented here are in contrast to those observations. CDK9 protein levels remained unchanged in human cells entering and progressing through the cell cycle from G(0), despite dramatic changes in SKP2 expression. CDK9 levels also remained unchanged in cells exiting from mitosis and progressing through the next cell cycle. Similarly, the levels of CDK9 protein did not change as cells exited the cell cycle and differentiated along various lineages. In keeping with these observations, the kinase activity associated with CDK9 was found to not be regulated during the cell cycle. We have also found that endogenous CDK9 is a very stable protein with a half-life (t(1/2)) of 4 to 7 h, depending on the cell type. In contrast, when CDK9 is overexpressed, it is not stabilized and is rapidly degraded, with a t(1/2) of less than 1 h, depending on the level of expression. Treatment of cells with proteasome inhibitors blocked the degradation of short-lived proteins, such as p27, but did not affect the expression of endogenous CDK9. Ectopic overexpression of SKP2 led to reduction of p27 protein levels but had no effect on the expression of endogenous CDK9. Finally, downregulation of endogenous SKP2 gene expression by interfering RNA had no effect on CDK9 protein levels, whereas p27 protein levels increased dramatically. Therefore, the SCF(SKP2) ubiquitin ligase does not regulate CDK9 expression in a cell cycle-dependent manner.

Developmental Regulation of the Proteolysis of the p35 Cyclin-Dependent Kinase 5 Activator by Phosphorylation

Cyclin-dependent kinase 5 (Cdk5), a cdc2-related kinase expressed in postmitotic neurons, is activated by association with a brain-specific activator, p35. It has been suggested that the conversion of p35 to p25 by the protease calpain is involved in neuronal cell death. However, p35 protein is turned over rapidly via proteasomal degradation in living neurons. In this study we show that the phosphorylation of p35 by Cdk5 suppresses the cleavage to p25 by calpain, whereas phosphorylation facilitates the proteasomal degradation of p35. The phosphorylation site in p35 that might be involved in preventing calpain cleavage was distinct from the phosphorylation site involved in facilitating proteasomal degradation. A phosphorylated form of p35 that was resistant to cleavage by calpain was more prevalent in the fetal brain, whereas the unphosphorylated form of p35 occurred in the adult brain. These results suggest that the phosphorylation of p35 serves as a protective mechanism that suppresses the generation of p25 in developing brains.

Gene Expression Profiling of Melanocytic Lesions

DNA microarrays, microscopic grids of DNA, can be used to assess gene expression within a particular cell or cell population. Since DNA from thousands of genes can be hybridized and analyzed in one experiment, researchers can globally characterize genes expressed in normal and various pathologic states. To accurately assess the differences between normal and pathologic states, one derives cDNA from control and diseased tissue specimens for genes expression profiling. For these reasons, microarray technology may be of particular interest to dermatopathologists and dermatologists interested in understanding cutaneous disease because these physicians have access to tissue specimens. In addition, microarray technology is an efficient way of identifying molecules expressed in a cell population; therefore, it can be used to search for unique immunohistologic markers. To this end, we have used microarray technology to define differences in the gene expression profile of nevi and melanomas. In this manuscript, we discuss the results of our study, which confirm previously known differences in gene expression between melanoma and nevi. While a few genes appear slightly overexpressed in nevi, a number of genes involved in regulating cell proliferation were upregulated in melanoma, such as cyclin D1, cdc2-related protein kinase, c-Myc binding protein, early growth response protein 1, and pleiotrophin. "Housekeeping" genes such as glyceraldehyde 3-phosphate dehydrogenase were expressed at similar levels in melanoma and nevi. Surprisingly, a majority of genes were expressed at similar levels in both nevi and melanoma. Based on this study, DNA microarray technology appears to be a valuable tool for identifying genes that may be specifically expressed in cutaneous lesions.

A novel TRH-PFTAIRE protein kinase 1 pathway in the cerebellum: subtractive hybridization analysis of TRH-deficient mice.

TRH has been reported to possess several neurophysiological actions in the brain. To gain insights into the molecular mechanisms underlying these effects, particularly in the cerebellum, we attempted to clone a cDNA that was regulated by TRH using TRH knockout mice and subtractive cDNA analysis. Over 100 clones obtained by subtractive hybridization analysis between the wild-type and TRH-1-cerebellum were analyzed. Four clones among them were identical and cdc2-related kinase (PFTAIRE protein kinase 1 (PFTK1)) cDNA, which was previously reported to be expressed only in the brain and testis. PFTK1 mRNA levels in the euthyroid TRH-1- cerebellum supplemented with thyroid hormone were significantly decreased compared with those in the wild-type. Induction of PFTK1 mRNA by TRH was also observed in a time- and dose-dependent manner in human medulloblastoma-derived HTB-185 cells that expressed TRH receptor subtype I mRNA. In addition, treatment of 8-Br-cGMP significantly increased PFTK1 mRNA levels, and a specific inhibitor of cGMP production, ODQ, completely blocked TRH-induced expression of PFTK1 mRNA. Furthermore, induction of PFrK1 mRNA by TRH was significantly inhibited by a NOS specific inhibitor, L-NAME, but not by a MEK inhibitor, PD98059 or a calcium channel inhibitor, nimodipine. These findings demonstrated, for the first time, a novel pathway between a neuropeptide and a cell cycle related peptide in the brain, and PFTK1 may be a key regulator for TRH action in t he cerebellum through t he NO-cGMP pathway.