Granulocyte-macrophage colony-stimulating factor abrogates transforming growth factor-beta 1-mediated cell cycle arrest by up-regulating cyclin D2/Cdk6

Beh?et's disease (BD) is a chronic multisystemic inflammatory disorder characterized mainly by recurrent oral and genital aphthous ulcerations and uveitis. Etiology and pathogenesis of BD remain unknown. T cell receptor (TCR) V alpha/V beta gene product expression as well as Jbeta gene segment expression in peripheral blood of BD patients were analysed to investigate the possible role of T lymphocytes in the etiopathogenesis of BD. Flow cytometry with 12 TCR V-specific MoAbs was used for TCRV analyses. Jbeta gene segment usage by T cell populations expressing certain V betas was determined by polymerase chain reaction (PCR) technique with V beta- and C beta-specific primers, Southern blotting of PCR products, and subsequent hybridization with radiolabelled Jbeta gene segment-specific probes. Although 13 of the 23 BD patients exhibited increases in expression of one or more TCR V-gene products, only expansions among the CD4+ T cell subset were significantly more frequent in BD patients (7/23) compared with healthy controls (0/15) (P = 0.019). Six out of eight cases followed for up to 20 months had at least one expansion correlated with disease activity. A strict preference for particular Jbeta gene segments implicating clonality was apparent in all analysed T cell expansions and correlated well with disease activity. These results suggest a possible involvement of antigen-specific T lymphocytes in the pathogenesis of BD.     

HER-2/c-erbB2 is phosphorylated by calmodulin-dependent protein kinase II on a single site in the cytoplasmic tail at threonine-1172

Calmodulin-dependent protein kinase II (Cam kinase II) is known to desensitise epidermal growth factor receptor (HER-1) tyrosine kinase activity by a process involving phosphorylation at serines 1046/47 in the cytoplasmic tail. We have developed an experimental system to investigate phosphorylation of the related HER-2/c-erbB2 proto-oncogene utilising purified Cam kinase II and recombinant glutathione-S-transferase (GST) fusion proteins. The cDNA for rat Cam kinase II-alpha was transfected into human embryonic kidney (HEK) 293 fibroblasts and the expressed protein purified to homogeneity by calmodulin-agarose affinity chromatography. A GST fusion protein comprising residues 1126-1255 of HER-2 was phosphorylated by purified Cam kinase II, in contrast to a GST protein comprising residues 1005-1125. Phosphoamino-acid analysis and site-directed mutagenesis indicated that HER-2 was phosphorylated on a single site at threonine-1172 which resides within a consensus Cam kinase II phosphorylation site (RAKT). HER-2 (threonine-1172-alanine), in the form of a ligand-inducible chimaera HER-1/2, was co-transfected into HEK-293 fibroblasts with a constitutively active form of Cam kinase II, followed by in vivo labelling of these cells with 32 P-orthophosphate. Immunoprecipitation of ligand-activated receptors followed by two-dimensional phosphopeptide mapping indicated that threonine-1172 in HER-2 is a newly identified in vivo site which can be hyper-phosphorylated by constitutively active Cam kinase II. In addition, when over-expressed in HEK-293 fibroblasts, HER-1/2 (threonine-1172-alanine) showed a defect in desensitisation and underwent a more sustained EGF-induced receptor autophosphorylation compared to wild-type HER-1/2.     

The kinase activity of c-Abl but not v-Abl is potentiated by direct interaction with RFXI, a protein that binds the enhancers of several viruses and cell-cycle regulated genes

c-Abl, the non-receptor tyrosine kinase is associated with EP, a DNA element found in promoters/enhancers of different viruses and cell-cycle regulated genes. EP-DNA binds RFXI, a member of a novel family of DNA-binding proteins that is conserved through evolution and in yeast, it controls differentiation and exit from the mitotic cycle to G0. EP-associated proteins are preferentially tyrosine phosphorylated and the associated c-Abl has strong tyrosine kinase activity. Here we investigated the molecular mechanism underlying this c-Abl kinase activity. We show that RFXI and c-Abl are in direct interaction, in vitro and in cell extracts, through the RFXI proline rich (PxxP) motif and the c-Abl SH3 domain. Remarkably, this interaction significantly potentiates c-Abl but not v-Abl auto-kinase activity. Collectively, we describe a novel mechanism of c-Abl recruitment to a defined DNA-cis element with its concomitant kinase activation. We propose that this mechanism may act to regulate cell-cycle control genes.     

The two-exon gene of the human forkhead transcription factor FREAC-2 (FKHL6) is located at 6p25.3

Subcutaneous metastases from clear cell endometrial carcinoma are an uncommon event and tumor implantations are rarely found with diagnostic imaging techniques. The nodular form is the most frequent type of subcutaneous metastasis from genital system tumors, even though plaque-like and infiltrative forms have also been reported. We report the first case of subcutaneous metastasis from clear cell endometrial carcinoma whose progression from the early nodular to the lymphangitic infiltrative form was studied with computed tomography (CT). Differential diagnostic problems are discussed.     

The cellular localization of the murine serine/arginine-rich protein kinase CLK2 is regulated by serine 141 autophosphorylation

Pre-mRNA splicing is catalyzed by a multitude of proteins including serine/arginine-rich (SR) proteins, which are thought to play a crucial role in the formation of spliceosomes and in the regulation of alternative splicing. SR proteins are highly phosphorylated, and their kinases are believed to regulate the recruitment of SR proteins from nuclear storage compartments known as speckles. Recently, a family of autophosphorylating kinases termed CLK (CDC2/CDC28-like kinases) was shown to phosphorylate SR proteins and to influence alternative splicing in overexpression systems. Here we used endogenous CLK2 protein to demonstrate that it displays different biochemical characteristics compared with its overexpressed protein and that it is differentially phosphorylated in vivo. Furthermore, CLK2 changed its nuclear localization upon treatment with the kinase inhibitor 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole. We have also identified a CLK2 autophosphorylation site, which is highly conserved among all CLK proteins, and we show by site-directed mutagenesis that its phosphorylation influences the subnuclear localization of CLK2. Our data suggest that CLK2 localization and possibly activity are influenced by a balance of CLK2 autophosphorylation and the regulation by CLK2 kinases and phosphatases.     

HiNF-D (CDP-cut/CDC2/cyclin A/pRB-complex) influences the timing of IRF-2-dependent cell cycle activation of human histone H4 gene transcription at the G1/S phase transition

The Bacillus stearothermophilus ribosomal protein S15 binds to the central domain of the 16 S rRNA inducing a conformational change in a three-way helical junction. To understand the nature of this conformational change, extended-helical junctions were prepared to examine the effects of S15 or Mg2+ binding on the relative helical orientation using native gel electrophoretic mobility and transient electric birefringence. The free junction is planar with approximately 120 degrees interhelical angles, whereas S15 and Mg2+ yield a junction conformation that remains planar in which two helices, 21 and 22, become colinear and the third, helix 20, forms a 60 degrees angle with respect to helix 22. This conformational change is thought to be important for directing the assembly of the central domain of the 30 S ribosomal subunit.     

Protein phosphatase 2A catalytic subunit is methyl-esterified at its carboxyl terminus by a novel methyltransferase

In eukaryotic cells a number of different proteins with important regulatory functions are reversibly methyl-esterified at carboxyl-terminal prenylcysteine residues. These proteins include the low molecular weight GTP-binding proteins, the gamma-subunit of the heterotrimeric G-proteins, and the nuclear lamins. The methylating enzymes that catalyze this type of carboxyl methylation reaction are integral membrane proteins, and the methylated protein products tend to be membrane-associated. Analyses of protein carboxyl methylation in a wide range of vertebrate tissues revealed a major carboxyl-methylated protein that was clearly distinct from those that are modified at prenylcysteine groups (Volker, C., Miller, R.A., McCleary, W.R., Rao, A., Poenie, M., Backer, J.M., and Stock, J.B. (1991) J. Biol. Chem. 266, 21515-21522). This M(r) = 36,000 protein is localized to the cytosol. Unlike the prenylcysteine methyltransferases, the enzyme that catalyzes the methylation of the 36-kDa protein is found in the cytosol. The 36-kDa methylated protein has been purified from bovine brain. Sequence analysis of several peptides clearly shows that the protein is the catalytic subunit of protein phosphatase 2A. A soluble 40-kDa methyltransferase that catalyzes the reaction has also been purified.