(13)(14)(15) and were suggested to be responsible for the kinase activity of TFIIH. Furthermore, a Saccharomyces cerevisiae M015-related kinase, KIN28, was identified as a p33 kinase subunit of yeast TFIIH (16). The TFIIH-associated KIN28 is included in the holo-TFIIH associated with transcription but can be separated from a form of TFIIH still functional in DNA repair (17).The C-terminal domain (CTD) of Pol II is a substrate for the TFIIH kinase (18), and one of the functions of TFIIH may be to regulate polymerase activity by modulating CTD phosphorylation. The exact role of CTD phosphorylation in transcription remains elusive. The CTD of polymerase entering the preinitiation complex (19) or stalled close to the promoter (20) is not phosphorylated, whereas the elongating polymerase is highly phosphorylated (21). In addition, CTD that is not phosphorylated interacts with the TATA-binding protein (TBP) component of TFIID (22) and with TFIIE (23), leading to the notion that phosphorylation of CTD would be critical for promoter clearance. On the other hand, kinase inhibitor studies have suggested that CTD phosphorylation is not required for basal transcription (24). Another model suggested by studies with yeast polymerase holoenzymes suggests thatThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.CTD phosphorylation might be involved in activated transcription (25,26). Here the role of M015 and cyclin H in CTD phosphorylation and the effects of this phosphorylation on transcription are discussed. MATERIALS AND METHODSAntibodies. A monoclonal antibody (mAb) against p62 (mAb 3c9) was provided by J.-M. Egly (Centre National de la Recherche Scientifique, Illkirch, France); the M015 polyclonal rabbit serum was raised against a 15-amino-acid Cterminal peptide; the cyclin H polyclonal rabbit serum was raised against a glutathione S-transferase (GST)-cyclin H fusion protein; the p89 antiserum was a polyclonal rabbit serum against a GST-p89-N-terminal fragment; and commercial preparations of the polyclonal rabbit CDK2 antibody (M2; Santa Cruz, Biotechnology, Santa Cruz, CA) and Myc mAb 9E10 (Cambridge Research Biochemicals) were used.Transfections. Fifteen micrograms of plasmids encoding wild-type or mutant MO15 cDNAs with C-terminal triple Myc-epitope tags (46) in pcDNA3 (Invitrogen) or 15 ,ug of pcDNA3 together with 15 ,g of an untagged cyclin H cDNA in pcDNA3 were transfected into human bone sarcoma U-2 OS cells by calcium phosphate coprecipitation. This particular cell line was used because of its high transfectability. Fortyeight hours after transfection, cells were washed in phosphatebuffered saline and lysed in ELB (150 mM NaCl/50 mM Hepes, pH 7.5/5 mM EDTA/0.1% Nonidet P-40 containing 10 Ag each of aprotinin and leupeptice per ml, 0.2 mM phenylmethylsulfonyl fluoride, 10 mM f3-glycerophosphate, and 5 mM NaF) for immunoprecipitation.Immunoblotting and Immunopre...

SummaryActin stress fiber assembly and contractility in nonmuscle motile cells requires phosphorylation of myosin regulatory light chain (MLC). Dephosphorylation and disassembly are mediated by MLC phosphatase, which is targeted to actin fibers by the association of its regulatory subunit MYPT1 with myosin phosphatase Rho-interacting protein (MRIP). In the present study, we identify the kinase NUAK2 as a second protein targeted by MRIP to actin fibers. Association of NUAK2 with MRIP increases MLC phosphorylation and promotes formation of stress fibers. This activity does not require the kinase activity of NUAK2 but is dependent on both MRIP and MYPT1, indicating that the NUAK2-MRIP association inhibits fiber disassembly and MYPT1-mediated MLC dephosphorylation. NUAK2 levels are strongly induced by stimuli increasing actomyosin fiber formation, and NUAK2 is required for fiber maintenance in exponentially growing cells, implicating NUAK2 in a positive-feedback loop regulating actin stress fibers independently of the MLC kinase Rho-associated protein kinase (ROCK). The identified MRIP-NUAK2 association reveals a novel mechanism for the maintenance of actin stress fibers through counteracting MYPT1 and, together with recent results, implicates the NUAK proteins as important regulators of the MLC phosphatase acting in both a kinase-dependent and kinase-independent manner.

Mutations in the tumor suppressor gene LKB1 are important in hereditary Peutz-Jeghers syndrome, as well as in sporadic cancers including lung and cervical cancer. LKB1 is a kinase-activating kinase, and a number of LKB1-dependent phosphorylation cascades regulate fundamental cellular and organismal processes in at least metabolism, polarity, cytoskeleton organization, and proliferation. Conditional targeting approaches are beginning to demonstrate the relevance and specificity of these signaling pathways in development and homeostasis of multiple organs. More than one of the pathways also appear to contribute to tumor growth following Lkb1 deficiencies based on a number of mouse tumor models. Lkb1-dependent activation of AMPK and subsequent inactivation of mammalian target of rapamycin signaling are implicated in several of the models, and other less well characterized pathways are also involved. Conditional targeting studies of Lkb1 also point an important role of LKB1 in epithelial-mesenchymal interactions, significantly expanding knowledge on the relevance of LKB1 in human disease.

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 G 1 /S transition, skp1-3f cells progress through S phase and instead display two distinct phenotypes. A fraction of the skp1-3f cells arrest in mitosis with high Cdc2 activity. Other skp1-3f cells as well as the skp1-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 of pof6 (for pombe F-box) as a Skp1-associated protein. Interestingly, cells deleted for the essential pof6 gene display a similar cell separation defect noted in skp1 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.