Dual-specificity phosphatase 14 (DUSP14, also known as MKP6) is a MAP kinase phosphatase that dephosphorylates JNK, ERK, and p38 in vitro. We recently reported that DUSP14 negatively regulates T-cell activation and immune responses by interfering activation of TAB1–TAK1 complex. However, the molecular mechanism that regulates the phosphatase activity of DUSP14 remains unclear. Here, we report the post-translational modification of DUSP14 by ubiquitination. Mass spectrometry and mutational analyses identified that DUSP14 was Lys63-linked ubiquitinated at lysine 103 residue. Furthermore, DUSP14 inducibly interacted with the E3 ligase TRAF2 during T-cell receptor (TCR) signaling; TRAF2 shRNA knockdown reduced the DUSP14 ubiquitination. We also show that ubiquitination of DUSP14 was required for its phosphatase activity during TCR signaling. Together, these findings reveal a novel mechanism by which TRAF2 mediates Lys63-linked ubiquitination of DUSP14, leading to DUSP14 activation in T cells.

After infection with unrelated viruses such as paramyxoviruses, orthomyxoviruses, poxviruses, and rhabdoviruses (1) mice generate cytolytic T lymphocytes (CTL) 1 that possess selective specificity for the sensitizing virus. However, after infection with different subtypes of serologically distinct influenza A viruses (2) at least more than one population of C T L can be demonstrated. Both cross-reactive and subtype-specific lymphocytes can be distinguished (3-12). These cross-reactive effector T ceils may lack the discriminatory capacity for influenza antigens shown by the B cell products, or they may recognize a shared antigenic determinant. Because the question of the nature of antigens recognized by T cells, as well as the structure and composition of the T cell receptor, is not yet clear, the orthomyxoviruses offer a biological relevant system for the analysis of T cell specificity. The subtypes of influenza A viruses differ with respect to the antigenicity of the envelope glycoproteins, hemagglutinin (HA) and neuraminidase (N). HA and N are inserted into the plasma membrane of cells during the budding process that results in virus release from the infected cells (13, 14). The internal virion components, the matrix (M) protein and the ribonucleoprotein (NP), of all influenza A viruses seem to be closely related antigenically. The cell-membrane expression of antigens is a prerequisite for T cell recognition (15-17). The work of several authors has therefore centered on the question of membrane expression of these antigens. M protein has now been detected on the cell membrane, and, consequently, it has been claimed that the cross-reactive T cell may recognize this protein (8, 18-22). This study aims to clarify the role of M protein in the C T L response. Target cells were prepared by two different methods. First by abortive infection of L-929 cells

The regulator of ubiquitous kinase (Ruk) protein, also known as CIN85 or SETA, is an adaptor-type protein belonging to the CD2AP/CMS family. It was found in complexes with many signaling proteins, including phosphoinositol (PtdIns) 3-kinase (EC 2.7.1.137), Cbl, GRB2, p130Cas and Crk. Functional analysis of these interactions, implicated Ruk in the regulation of apoptosis, receptor endocytosis and cytoskeletal rearrangements. We have recently demonstrated that overexpression of Ruk induces apoptotic death in neurons, which could be reversed by activated forms of PtdIns 3-kinase and PKB/Akt. Furthermore, Ruk was shown to be a negative regulator of PtdIns 3-kinase activity through binding to its P85 regulatory subunit [Gout, I., Middleton, G., Adu, J., Ninkina, N. N., Drobot, L. B., Filonenko, V., Matsuka, G., Davies, A.M., Waterfield, M. & Buchman, V. L. (2000) Embo J.19, 4015-4025]. Here, we report for the first time, that all three isoforms of Ruk (L, M and S) are ubiquitinated. Specific interaction between the E3 ubiquitin ligase Cbl and all three Ruk isoforms was demonstrated by coexpression studies in Hek293 cells. The interaction of Ruk M and S isoforms with Cbl was found to be mediated via heterodimerization with Ruk L. The use of proteosomal and lysosomal inhibitors clearly indicated that ubiquitination of Ruk L does not lead to its degradation. Based on this study, we propose a possible mechanism for the regulation of Ruk function by ubiquitination.

Class IA phosphatidylinositol 3‐kinase (PI 3‐kinase) is a key component of important intracellular signalling cascades. We have identified an adaptor protein, Rukl, which forms complexes with the PI 3‐kinase holoenzyme in vitro and in vivo. This interaction involves the proline‐rich region of Ruk and the SH3 domain of the p85α regulatory subunit of the class IA PI 3‐kinase. In contrast to many other adaptor proteins that activate PI 3‐kinase, interaction with Rukl substantially inhibits the lipid kinase activity of the enzyme. Overexpression of Rukl in cultured primary neurons induces apoptosis, an effect that could be reversed by co‐expression of constitutively activated forms of the p110α catalytic subunit of PI 3‐kinase or its downstream effector PKB/Akt. Our data provide evidence for the existence of a negative regulator of the PI 3‐kinase signalling pathway that is essential for maintaining cellular homeostasis. Structural similarities between Ruk, CIN85 and CD2AP/CMS suggest that these proteins form a novel family of adaptor molecules that are involved in various intracellular signalling pathways.

© 1999, National Academy of Sciences of Ukraine. All rights reserved. A novel ribosomal S6 kinase, termed p70S6 kinase β, has been recently identified. It is highly homologous to known p70/p85 S6 kinase (p70S6 kinase α) in catalytic, kinase extension and auto-inhibitory domains, but differs significantly in the regulatory N- and C-terminal regions. Here we report the kinetics of serum-induced activation of cytoplasmic isoforms of p70S6 kinase α and β (p70α2 and p70β2) in transiently transfected HEK293. Both kinases exhibit biphasic activation upon serum stimulation, but differ in the appearance of peaks of their S6 specific activity. We also found that p70α2 and p70β2, when overexpressed in HEK293 cells, are sensitive to the immunosuppressant rapamycin. However, p70β2 kinase is more sensitive to low concentrations of rapamycin, when compared with p70α2. At the same time, high concentrations of rapamycin are inhibitory to a higher extent towards p70α2, than p70β2 kinase. Taken together, our results indicate that p70α2 kinase is activated earlier in response to serum and is less sensitive to high concentration of rapamycin, in comparison to p70α2. These differences indicate the existence of alternative signalling mechanisms, involved in the regulation of p70β2 kinase.

Phosphoinositide 3-kinases (PI3-kinases) have been shown to be recruited to cell surface receptor signal complexes whose formation is triggered by growth factors, cytokines and other ligands. PI3-kinases are also involved in protein sorting phenomena. A number of PI3-kinase isotypes have been characterised in several laboratories. Here the relations between the PI3-kinases, PI4-kinases and PI5-kinases and other potential phosphoinositide kinases are analysed. A study of the relation of structure to function for sequence motifs defined through the use of homology searches and protein modelling techniques is described and used to assign the family of phosphoinositide kinases to subgroups.

The pleckstrin homology (PH) domain is a conserved module present in many signal transducing and cytoskeletal proteins. Here we report the 2.8 Angstrom crystal structure of the PH domain from dynamin. This domain consists of seven beta-strands forming two roughly orthogonal antiparallel beta-sheets terminating with an amphipathic alpha-helix. The structure also reveals a non-covalent dimeric association of the PH domain and a hydrophobic pocket surrounded by a charged rim. The dynamin PH domain structure is discussed in relation to its potential role in mediating interactions between proteins.

The N‐terminal src‐homology 2 domain of the p85α subunit of phosphatidylinositol 3′ kinase (SH2‐N) binds specifically to phosphotyrosine‐containing sequences. Notably, it recognizes phosphorylated Tyr 751 within the kinase insert of the cytoplasmic domain of the activated βPDGF receptor. A titration of a synthetic 12‐residue phosphopeptide (ESVDY*VPMLDMK) into a solution of the SH2‐N domain was monitored using heteronuclear 2D and 3D NMR spectroscopy. 2D‐[15N‐1H] heteronuclear single‐quantum correlation (HSQC) experiments were performed at each point of the titration to follow changes in both 15N and 1H chemical shifts in NH groups. When mapped onto the solution structure of the SH2‐N domain, these changes indicate a peptide‐binding surface on the protein. Line shape analysis of 1D profiles of individual [15N‐1H]‐HSQC peaks at each point of the titration suggests a kinetic exchange model involving at least 2 steps. To characterize changes in the internal dynamics of the domain, the magnitude of the [15N‐1H] heteronuclear NOE for the backbone amide of each residue was determined for the SH2‐N domain with and without bound peptide. These data indicate that, on a nanosecond timescale, there is no significant change in the mobility of either loops or regions of secondary structure. A mode of peptide binding that involves little conformational change except in the residues directly involved in the 2 binding pockets of the p85α SH2‐N domain is suggested by this study.

Two species of PtdIns 4-kinase with molecular masses of 50 kDa and 45 kDa were detected in human erythrocyte membranes using SDS/PAGE. These enzymes were purified to near homogeneity and found to display very similar enzymatic characteristics. The purification scheme consisted of solubilization from erythrocyte membranes in the presence of Triton X-100, followed by Cibacron-blue-Sephadex, phosphocellulose and Mono Q anion-exchange chromatography. The final step in the purification protocol was preparative SDS/PAGE, followed by electroelution and renaturation of the enzyme. This procedure afforded an about 4000-fold purification of the enzyme from erythrocyte membranes. Characterization of the [32P]PtdInsP products formed by the purified PtdIns kinases indicated that these enzymes specifically phosphorylated the D-4 position of the inositol ring. The Km values of both PtdIns 4-kinase species for PtdIns and ATP were found to be 0.2 mM and 0.1 mM, respectively. The enzymes are both activated by Mg2+, and inhibited by Ca2+ and by adenosine. The potential importance of these effectors for the regulation of PtdIns phosphorylation in cells is discussed.

The human epidermal growth factor receptor (EGFR) proto-oncogene is shown to span 110 kb of DNA divided into 26 exons. Analysis of sequences surrounding exon 1 reveals a highly CG rich region which promotes transcription. The activity of the EGF receptor promoter can be modulated by E1A protein and receptor RNA levels increased by stimulation with phorbol ester or fetal calf serum. Promoter activity is assayed by linkage to the chloramphenicol acetyl transferase gene and transfection in three cell lines, with quantitation of plasmid DNA uptake by isolation of a Hirt supernatent from each transfection. Deletion analysis of the CG rich promoter region of the gene and construction of chimeric EGFR/SV40 promoters are used to demonstrate positive transcription elements located both within exon 1 and 5' to the start of transcription. Negative regulation of transcription by sequences within a -140 to +80 region is suggested. Cotransfection experiments suggest a requirement for the interaction of DNA binding protein Sp1 for maximal activity. Finally, derepression of a positive regulatory sequence located in exon 1 during cotransfection experiments is shown. Results are discussed in reference to the multilevel regulation of EGF receptor expression.

The mechanism by which the estrogen receptor and other steroid hormone receptors regulate gene expression in eukaryotic cells is not well understood. In this study, a complementary DNA clone containing the entire translated portion of the messenger RNA for the estrogen receptor from MCF-7 human breast cancer cells was sequenced and then expressed in Chinese hamster ovary (CHO-K1) cells to give a functional protein. An open reading frame of 1785 nucleotides in the complementary DNA corresponded to a polypeptide of 595 amino acids and a molecular weight of 66,200, which is in good agreement with published molecular weight values of 65,000 to 70,000 for the estrogen receptor. Homogenates of transformed Chinese hamster ovary cells contained a protein that bound [ 3 H]estradiol and sedimented as a 4S complex in salt-containing sucrose gradients and as an 8 to 9S complex in the absence of salt. Interaction of this receptor-[ 3 H]estradiol complex with a monoclonal antibody that is specific for primate ER confirms the identity of the expressed complementary DNA as human estrogen receptor. Amino acid sequence comparisons revealed significant regional homology among the human estrogen receptor, the human glucocorticoid receptor, and the putative v- erb A oncogene product. This suggests that steroid receptor genes and the avian erythroblastosis viral oncogene are derived from a common primordial gene. The homologous region, which is rich in cysteine, lysine, and arginine, may represent the DNA-binding domain of these proteins.

Poly(A)+ RNA isolated from the human breast cancer cell line MCF-7 was fractionated by sucrose gradient centrifugation and fractions enriched in estrogen receptor (ER) mRNA were used to prepare randomly primed cDNA libraries in the lambda gt10 and lambda gt11 vectors. Clones corresponding to ER sequence were isolated from both libraries after screening with either ER monoclonal antibodies (lambda gt11) or synthetic oligonucleotide probes designed from two peptide sequences of purified ER (lambda gt10). Five cDNA clones were isolated by antibody screening and five were isolated after screening with synthetic oligonucleotides. The two largest ER cDNA clones, lambda OR3 (1.3 kilobase pairs) and lambda OR8 (2.1 kilobase pairs), isolated by using antibodies and oligonucleotides, respectively, were able to enrich selectively for ER mRNA by hybrid-selection. Furthermore, lambda OR8 contains the DNA sequence expected from the two ER peptides and crosshybridizes with each of the other ER cDNA clones. These results demonstrate that the clones isolated correspond to the ER mRNA sequence. Use of lambda OR8 as a hybridization probe revealed a single poly(A)+ RNA band of approximately equal to 6.2 kilobase pairs in the ER-containing human breast cancer cell lines MCF-7 and T47D. In contrast, no hybridization was seen in the human ER-negative cell line HeLa. The same probe hybridizes to a chicken gene that is expressed in oviduct tissue as a 7.5-kilobase-pair poly(A)+ RNA.