Mutations in LKB1 lead to Peutz–Jeghers syndrome (PJS). However, only a subset of PJS patients harbours LKB1 mutations. We performed a mutation analysis of three genes encoding novel LKB1-interacting proteins, BRG1 , STRADα , and MO25α , in 28 LKB1 -negative PJS patients. No disease-causing mutations were detected in the studied genes in PJS patients from different European populations.

We recently demonstrated that the LKB1 tumour suppressor kinase, in complex with the pseudokinase STRAD and the scaffolding protein MO25, phosphorylates and activates AMP-activated protein kinase (AMPK). A total of 12 human kinases (NUAK1, NUAK2, BRSK1, BRSK2, QIK, QSK, SIK, MARK1, MARK2, MARK3, MARK4 and MELK) are related to AMPK. Here we demonstrate that LKB1 can phosphorylate the T-loop of all the members of this subfamily, apart from MELK, increasing their activity >50-fold. LKB1 catalytic activity and the presence of MO25 and STRAD are required for activation. Mutation of the T-loop Thr phosphorylated by LKB1 to Ala prevented activation, while mutation to glutamate produced active forms of many of the AMPK-related kinases. Activities of endogenous NUAK2, QIK, QSK, SIK, MARK1, MARK2/3 and MARK4 were markedly reduced in LKB1-deficient cells. Neither LKB1 activity nor that of AMPK-related kinases was stimulated by phenformin or AICAR, which activate AMPK. Our results show that LKB1 functions as a master upstream protein kinase, regulating AMPK-related kinases as well as AMPK. Between them, these kinases may mediate the physiological effects of LKB1, including its tumour suppressor function.

BACKGROUND & AIMSPeutz-Jeghers syndrome (PJS) is typically manifested as severe gastrointestinal polyposis. Polyps in PJS patients and in Lkb1(+/-) mice that model PJS polyposis are frequently characterized by elevated cyclooxygenase-2 (COX-2). This study was designed to determine whether COX-2 inhibition would reduce tumor burden in Lkb1(+/-) mice or Peutz-Jeghers patients.METHODSGenetic interactions between Cox-2 and Lkb1 in polyp formation were analyzed in mice with combined deficiencies in these genes. Pharmacologic inhibition of COX-2 was achieved by supplementing the diet of Lkb1(+/-) mice with 1500 ppm celecoxib between 3.5-10 and 6.5-10 months. In PJS patients, COX-2 was inhibited with a daily dose of 2 x 200 mg celecoxib for 6 months.RESULTSTotal polyp burden in Lkb1(+/-) mice was significantly reduced in a Cox-2(+/-) (53%) and in a Cox-2(-/-) (54%) background. Celecoxib treatment initiating before polyposis (3.5-10 months) led to a dramatic reduction in tumor burden (86%) and was associated with decreased vascularity of the polyps. Late treatment (6.5-10 months) also led to a significant reduction in large polyps. In a pilot clinical study, a subset of PJS patients (2/6) responded favorably to celecoxib with reduced gastric polyposis.CONCLUSIONSThese data establish a role for COX-2 in promoting Peutz-Jeghers polyposis and suggest that COX-2 chemoprevention may prove beneficial in the treatment of PJS.

Primary effusion lymphomas (PELs) represent a unique non-Hodgkin lymphoma that is consistently infected by Kaposi sarcoma herpesvirus (KSHV). PEL cells express high levels of the cell cycle inhibitor p27(KIP1) and yet proliferate actively. KSHV genome encodes a viral cyclin homolog, v-cyclin, which has previously been implicated in down-regulation of p27(KIP1) levels. To address how PEL cells can tolerate high p27(KIP1) levels, we investigated functional interactions between v-cyclin and p27(KIP1) using PEL-derived cell lines as a model system. Here we demonstrate that v-cyclin and p27(KIP1) stably associate in PEL cells in vivo suggesting an attractive model by which p27(KIP1) is inactivated in the actively proliferating PEL cells. Moreover, we show that v-cyclin and cyclin-dependent kinase 6 (CDK6) form an active kinase without p27(KIP1) and that CDK6 is the in vivo catalytic subunit of v-cyclin in PEL cells. These findings suggest that KSHV may promote oncogenesis in PEL by expressing v-cyclin, which both overrides negative cell cycle controls present in the PEL precursor cells and induces a strong proliferative signal via CDK6 kinase activity.

The fission yeast Schizosaccharomyces pombe has proved an important model system for cross-species comparative studies of many fundamental processes in the eukaryotic cell, such as cell cycle control and DNA replication. The RNA polymerase II transcription machinery is, however, still relatively poorly understood in S. pombe, partially due to the absence of a reconstituted in vitro transcription system. We have now purified S. pombe RNA polymerase II and its general initiation factors TFIIB, TFIIF, TFIIE, and TFIIH to near homogeneity. These factors enable RNA polymerase II to initiate transcription from the S. pombe alcohol dehydrogenase promoter (adh1p) when combined with Saccharomyces cerevisiae TATA-binding protein. We use our reconstituted system to examine effects of Mediator on basal transcription in vitro. S. pombe Mediator exists in two distinct forms, a free form, which contains the spSrb8, spTrap240, spSrb10, and spSrb11 subunits, and a smaller form, which lacks these four subunits and associates with RNA polymerase II to form a holoenzyme. We find that spSrb8/spTrap240/spSrb10/spSrb11 containing Mediator repress basal transcription, whereas Mediator lacking these subunits has a stimulatory effect on transcription. Our findings thus demonstrate that the spSrb8/spTrap240/spSrb10/spSrb11 subcomplex governs the ability of Mediator to stimulate or repress basal transcription in vitro.

Here we report functional characterization of the essential fission yeast Skp1 homologue. We have created a conditional allele of skp1 (skp1–3f) mimicking the mutation in the budding yeast skp1–3 allele. Although budding yeast skp1–3 arrests at the G1/S transition, skp1–3f cells progress through S phase and instead display two distinct phenotypes. A fraction of theskp1–3f cells arrest in mitosis with high Cdc2 activity. Other skp1–3f cells as well as theskp1-deleted cells accumulate abnormal thick septa leading to defects in cell separation. Subsequent identification of 16 fission yeast F-box proteins led to identification of the product ofpof6 (for pombe F-box) as a Skp1-associated protein. Interestingly, cells deleted for the essentialpof6 gene display a similar cell separation defect noted inskp1 mutants, and Pof6 localizes to septa and cell tips. Purification of Pof6 demonstrates association of Skp1, whereas the Pcu1 cullin was absent from the complex. These findings reveal an essential non-Skp1-Cdc53/Cullin-F-box protein function for the fission yeast Skp1 homologue and the F-box protein Pof6 in cell separation.

The histidine triad (HIT) protein Hint has been found to associate with mammalian Cdk7, as well as to interact both physically and genetically with the budding yeast Cdk7 homologue Kin28. To study the function of Hint and to explore its possible role in modulating Cdk7 activity in vivo, we have characterized the expression pattern of murine Hint and generated Hint-deficient (Hint(-/-)) mice. Hint was widely expressed during mouse development, with pronounced expression in several neuronal ganglia, epithelia, hearts, and testes from embryonic day 15 onward. Despite this widespread expression, disruption of Hint did not impair murine development. Moreover, Hint-deficient mice had a normal life span and were apparently healthy. Histological examination of tissues with high Hint expression in wild-type animals did not show signs of abnormal pathology in Hint(-/-) mice. Functional redundancy within the HIT family was addressed by crossing Hint(-/-) mice with mice lacking the related HIT protein, Fhit, and by assaying the expression levels of the HIT protein gene family members Hint2 and Hint3 in Hint(+/+) and Hint(-/-) tissues. Finally, Cdk7 kinase activity and cell cycle kinetics were found to be comparable in wild-type and Hint(-/-) mouse embryonic fibroblasts, suggesting that Hint may not be a key regulator of Cdk7 activity.

Hypertrophy allows the heart to adapt to increased stress, but eventually dysfunction and dilatation ensue. Why adaptation is incomplete and which molecular mechanisms cause functional deterioration are unclear. The global increase in RNA content in hypertrophy is accompanied by activation of two cyclin-dependent kinases (Cdks) that phosphorylate the RNA polymerase II C-terminal domain (CTD): Cdk9/cyclin T and Cdk7/cyclin H/menage-a-trois (MAT1). Cdk9 is activated by diverse hypertrophic stimuli through dissociation of 7SK small nuclear RNA (snRNA), an intrinsic inhibitor, and is required for hypertrophic growth in culture; Cdk7 activity was dispensable. Here, we report the increased activity of both Cdk7 and Cdk9 in human heart failure (5 of 7 patients with dilated cardiomyopathy; DCM). To clarify the role of CTD kinases more conclusively, we created complementary gain- and loss-of-function models in mice. Cardiac-specific expression of cyclin T1 (alpha MHC-cyc T1) induced Cdk9 activity, with proportional hypertrophic growth. Cardiac-specific expression of dominant-negative Cdk9 (alpha MHC-dnCdk9) did not impair baseline growth or function. We subjected the dnCdk9 and cyc T1 mice to pressure overload by transverse aortic constriction (TAC) or to mating with alpha MHC-Gq mice, models of compensated hypertrophy. Cyc T1 induced Cdk9 activity, impaired LV contractility, and conferred rapid lethality in both settings. Cyc T1×Gq double transgenic mice showed DCM and died by 3–4 wks; myofilament disarray, fibrosis and cardiomyocyte apoptosis were overt. Although neither banding nor cyc T1 impaired LV contractility singly, banding impaired LV function in cyc T1 mice (peak aortic flow velocity: before, 109.6±2.2, after, 67.2±3.7; p<0.05) and increased mortality (non-transgenic, 0%; cyc T1, 57.1%). By microarray comparisons, cyc T1 caused down-regulation of nuclear-encoded respiratory chain genes, carnitine palmitoyl transferase, and Mn superoxide dismutase, suggesting that reduced ATP production and increased reactive oxygen species may contribute to the observed exacerbation of contractility. dnCdk9 blocked the increase in Cdk9 activity expected from pressure overload and Gq; baseline activity was increased paradoxically, attributed to dissociation of the 7SK inhibitor. But, dnCdk9 mice tolerated hypertrophic signals poorly: both stresses caused ventricular dilatation and death. Cardiac-restricted deletion of MAT1 caused DCM. MAT1-deficient hearts appeared normal initially, but LV dilatation and severe systolic dysfunction occurred, with severe apoptosis and death of heart failure by 6 wks. In conclusion: (1) CTD kinase activity was increased in human heart failure. (2) After hypertrophic signals, excess Cdk9 activity and a complete block to increased Cdk9 activity each were adverse in mice. (3) Cardiac-specific deletion of MAT1 revealed an essential function of MAT1 in myocyte survival after birth. (4) Partial inhibition of Cdk9 might be beneficial to prevent unfavorable effects of Cdk9 in heart failure.

Germline mutations of the LKB1 tumor suppressor gene lead to Peutz-Jeghers syndrome (PJS), with a predisposition to cancer. LKB1 encodes for a nuclear and cytoplasmic serine/threonine kinase, which is inactivated by mutations observed in PJS patients. Restoring LKB1 activity into cancer cell lines defective for its expression results in a G(1) cell cycle arrest. Here we have investigated molecular mechanisms leading to this arrest. Reintroduced active LKB1 was cytoplasmic and nuclear, whereas most kinase-defective PJS mutants of LKB1 localized predominantly to the nucleus. Moreover, when LKB1 was forced to remain cytoplasmic through disruption of the nuclear localization signal, it retained full growth suppression activity in a kinase-dependent manner. LKB1-mediated G(1) arrest was found to be bypassed by co-expression of the G(1) cyclins cyclin D1 and cyclin E. In addition, the protein levels of the CDK inhibitor p21(WAF1/CIP1) and p21 promoter activity were specifically upregulated in LKB1-transfected cells. Both the growth arrest and the induction of the p21 promoter were found to be p53-dependent. These results suggest that growth suppression by LKB1 is mediated through signaling of cytoplasmic LKB1 to induce p21 through a p53-dependent mechanism.

In this report we have characterized a novel, ubiquitously expressed kinase, Clik1, that is predominantly nuclear and undergoes autophosphorylation. Yeast two-hybrid analysis indicated a highly specific association between Clik1 and CLP-36, which was identified in 36 out of 37 Clik1-interacting clones. CLP-36 is a PDZ-LIM protein that localizes to actin stress fibers in nonmuscle cells and associates with alpha-actinin via its PDZ-domain. The association of CLP-36 with Clik1, in turn, is mediated by the C-terminal part of CLP-36 containing the LIM domain, and association was not noted with the closely related ALP PDZ-LIM protein. Interestingly, the association with CLP-36 led to relocalization of the otherwise nuclear Clik1 kinase to actin stress fibers, where it disrupted the periodic staining pattern of CLP-36. Taken together these results establish the CLP-36 PDZ-LIM protein as an adapter, recruiting the Clik1 kinase to actin stress fibers in nonmuscle cells, and suggest that Clik1 represents a novel regulator of actin stress fibers.

The mammalian Mat1 protein has been implicated in cell cycle regulation as part of the Cdk activating kinase (CAK), and in regulation of transcription as a subunit of transcription factor TFIIH. To address the role of Mat1 in vivo, we have used a Cre/loxP system to conditionally ablate Mat1 in adult mitotic and post-mitotic lineages. We found that the mitotic cells of the germ lineage died rapidly upon disruption of Mat1 indicating an absolute requirement of Mat1 in these cells. By contrast, post-mitotic myelinating Schwann cells were able to attain a mature myelinated phenotype in the absence of Mat1. Moreover, mutant animals did not show morphological or physiological signs of Schwann cell dysfunction into early adulthood. Beyond 3 months of age, however, myelinated Schwann cells in the sciatic nerves acquired a severe hypomyelinating morphology with alterations ranging from cells undergoing degeneration to completely denuded axons. This phenotype was coupled to extensive proliferation and remyelination that our evidence suggests was undertaken by the non-myelinated Schwann cell pool. These results indicate that Mat1 is not essential for the transcriptional program underlying the myelination of peripheral axons by Schwann cells and suggest that the function of Mat1 in RNA polymerase II-mediated transcription in these cells is regulatory rather than essential.

Hantaviruses cause two severe diseases, haemorrhagic fever with renal syndrome in Eurasia and hantavirus pulmonary syndrome in the Americas. To understand more about the molecular mechanisms that lead to these diseases, the associations of Puumala virus nucleocapsid protein (PUUV-N) with cellular proteins were studied by yeast two-hybrid screening. Daxx, known as an apoptosis enhancer, was identified from a HeLa cDNA library and its interaction with PUUV-N was confirmed by GST pull-down assay, co-immunoprecipitation and co-localization studies. Furthermore, domains of interaction were mapped to the carboxyl-terminal region of 142 amino acids in Daxx and the carboxyl-terminal 57 residues in PUUV-N, respectively. In pepscan assays, the binding sites of Daxx to PUUV-N were mapped further to two lysine-rich regions, of which one overlaps the sequence of the predicted nuclear localization signal of Daxx. These data suggest a direct link between host cell machinery and a hantavirus structural component.

Lymphatic vessels are essential for fluid homeostasis, immune surveillance and fat adsorption, and also serve as a major route for tumor metastasis in many types of cancer. We found that isolated human primary lymphatic and blood vascular endothelial cells (LECs and BECs, respectively) show interesting differences in gene expression relevant for their distinct functions in vivo. Although these phenotypes are stable in vitro and in vivo, overexpression of the homeobox transcription factor Prox-1 in the BECs was capable of inducing LEC-specific gene transcription in the BECs, and, surprisingly, Prox-1 suppressed the expression of approximately 40% of the BEC-specific genes. Prox-1 did not have global effects on the expression of LEC-specific genes in other cell types, except that it up-regulated cyclin E1 and E2 mRNAs and activated the cyclin e promoter in various cell types. These data suggest that Prox-1 acts as a cell proliferation inducer and a fate determination factor for the LECs. Furthermore, the data provide insights into the phenotypic diversity of endothelial cells and into the possibility of transcriptional reprogramming of differentiated endothelial cells.

Transforming growth factor-beta (TGF-beta) signaling is mediated from serine/threonine kinase receptors to transcriptional responses via Smad proteins. Here comparison of mRNA expression of Smad3–7 in mouse embryos (E9-E15) revealed developmentally regulated distinct expression patterns for Smad3, 4, 6, and 7. Smad3 was prominently expressed in the differentiating (from E10) central nervous system, but also in developing bones, branchial arches and epithelium of various tissues. Smad4 mostly showed ubiquitous expression, but in E15 embryos, a pronounced signal appeared in epithelial crypts of the gut. Inhibitory Smad6 and Smad7 were coexpressed at high levels in developing cardiovascular system from the earliest stages studied. In contrast, Smad6 was selectively expressed at high levels, e.g. in intramembranous bone whereas Smad7 was prominent in seminiferous tubules of the testis, demonstrating distinct expression of these genes in non-cardiovascular tissues.

The LKB1 tumor suppressor gene, mutated in Peutz-Jeghers syndrome, encodes a serine/threonine kinase of unknown function. Here we show that mice with a targeted disruption of Lkb1 die at midgestation, with the embryos showing neural tube defects, mesenchymal cell death, and vascular abnormalities. Extraembryonic development was also severely affected; the mutant placentas exhibited defective labyrinth layer development and the fetal vessels failed to invade the placenta. These phenotypes were associated with tissue-specific deregulation of vascular endothelial growth factor ( VEGF ) expression, including a marked increase in the amount of VEGF messenger RNA. Moreover, VEGF production in cultured Lkb1 −/− fibroblasts was elevated in both normoxic and hypoxic conditions. These findings place Lkb1 in the VEGF signaling pathway and suggest that the vascular defects accompanying Lkb1 loss are mediated at least in part by VEGF.

In normal cells, activation of cyclin-dependent kinases (cdks) requires binding to a cyclin and phosphorylation by the cdk-activating kinase (CAK). The Kaposi's sarcoma-associated herpesvirus encodes a protein with similarity to D-type cyclins. This KSHV-cyclin activates CDK6, alters its substrate specificity, and renders CDK6 insensitive to inhibition by the cdk inhibitor p16(INK4a). Here we investigate the regulation of the CDK6/KSHV-cyclin kinase with the use of purified proteins and a cell-based assay. We find that KSHV-cyclin can activate CDK6 independent of phosphorylation by CAK in vitro. In addition, CAK phosphorylation decreased the p16(INK4a) sensitivity of CDK6/KSHV-cyclin complexes. In cells, expression of CDK6 or to a lesser degree of a nonphosphorylatable CDK6(T177A) together with KSHV-cyclin induced apoptosis, indicating that CDK6 activation by KSHV-cyclin can proceed in the absence of phosphorylation by CAK in vivo. Coexpression of p16 partially protected cells from cell death. p16 and KSHV-cyclin can form a ternary complex with CDK6 that can be detected by binding assays as well as by conformational changes in CDK6. The Kaposi's sarcoma-associated herpesvirus has adopted a clever strategy to render cell cycle progression independent of mitogenic signals, cdk inhibition, or phosphorylation by CAK.