CIS associates with the interleukin-2 receptor beta chain and inhibits interleukin-2-dependent signaling.

CIS is a cytokine-induced SH2-containing protein that was originally cloned as an interleukin (IL)-3-inducible gene. CIS is known to associate with the IL-3 receptor beta chain and erythropoietin receptor and to inhibit signaling mediated by IL-3 and erythropoietin. We now demonstrate that CIS also interacts with the IL-2 receptor beta chain (IL-2Rbeta). This interaction requires the A region of IL-2Rbeta (residues 313-382), which also mediates the association of IL-2Rbeta with Lck and Jak3. Correspondingly, CIS inhibits functions associated with both of these kinases: Lck-mediated phosphorylation of IL-2Rbeta and IL-2-mediated activation of Stat5. Thus, we demonstrate that CIS can negatively control at least two independent IL-2 signaling pathways. Although a functional SH2 binding domain of CIS was not required for its interaction with IL-2Rbeta in vitro, its phosphotyrosine binding capability was essential for the inhibitory action of CIS. On this basis, we have generated a mutant form of CIS protein with an altered SH2 domain that acts as a dominant negative and should prove useful in further understanding CIS action.     

Identification of key amino acids of the mouse mammary tumor virus superantigen involved in the specific interaction with T-cell receptor V(beta) domains.

Mouse mammary tumor virus (MMTV) is a retrovirus encoding a superantigen that is recognized in association with major histocompatibility complex class II by the variable region of the beta chain (V(beta)) of the T-cell receptor. The C-terminal 30 to 40 amino acids of the superantigen of different MMTVs display high sequence variability that correlates with the recognition of particular T-cell receptor V(beta) chains. Interestingly, MMTV(SIM) and mtv-8 superantigens are highly homologous but have nonoverlapping T-cell receptor V(beta) specificities. To determine the importance of these few differences for specific V(beta) interaction, we studied superantigen responses in mice to chimeric and mutant MMTV(SIM) and mtv-8 superantigens expressed by recombinant vaccinia viruses. We show that only a few changes (two to six residues) within the C terminus are necessary to modify superantigen recognition by specific V(beta)s. Thus, the introduction of the MMTV(SIM) residues 314-315 into the mtv-8 superantigen greatly decreased its V(beta)12 reactivity without gain of MMTV(SIM)-specific function. The introduction of MMTV(SIM)-specific residues 289 to 295, however, induced a recognition pattern that was a mixture of MMTV(SIM)- and mtv-8-specific V(beta) reactivities: both weak MMTV(SIM)-specific V(beta)4 and full mtv-8-specific V(beta)11 recognition were observed while V(beta)12 interaction was lost. The combination of the two MMTV(SIM)-specific regions in the mtv-8 superantigen established normal MMTV(SIM)-specific V(beta)4 reactivity and completely abolished mtv-8-specific V(beta)5, -11, and -12 interactions. These new functional superantigens with mixed V(beta) recognition patterns allowed us to precisely delineate sites relevant for molecular interactions between the SIM or mtv-8 superantigen and the T-cell receptor V(beta) domain within the 30 C-terminal residues of the viral superantigen.     

An affinity scale for the interaction of amino acid residues with nucleic acids

The affinity of amino acid residues to nucleic acids is probed by measurements of melting temperatures t_m for the helix–coil transition at various concentrations of amino acid amides. The increase of t_m on addition of ligand is described by the equation t_m = t*_m + αlog(1+K_tc_λ), where t*_m is the melting temperature in the absence of ligand, c_λ the ligand concentration, and K_t the “t_m-onset” constant, which is analogous to an equilibrium constant. It is shown that K_t is closely related to the affinity of the ligands to the double helix, whereas the slope α mainly reflects the preference of the ligand binding to the helix versus the coil form. In the case of the amino acid amides, α is found to be virtually independent of the nature of the side chain with few exceptions, e. g., aromatic amides. The t_m-onset constant, however, strongly depends on the nature of the amino acid side chain. For simple aliphatic amino acids, the relative free energy of binding decreases with increasing hydrophobic free energy, e.g., a high affinity is found for Gly-amide and a low affinity for Leu-amide. This relation is modified by functional groups like OH in Ser-amide. The helices poly[d(A-T)], ploy[d(I-C)]. and poly[d(A-C)]·poly[d(G-T)] exhibit similar affinity scales with relatively small variations. Our results demonstrate that the hydrophilic character of double helices at their surface disfavors binding of hydrophobic ligands unless special contacts can be formed. From our results we establish an affinity scale for the binding of amino acids to double helices.     

Physical interaction between interleukin-12 receptor beta 2 subunit and Jak2 tyrosine kinase: Jak2 associates with cytoplasmic membrane-proximal region of interleukin-12 receptor beta 2 via amino-terminus

IL-12 is a heterodimeric cytokine, composed of p40 and p35 subunits, that exerts its biological effects by binding to specific cell surface receptors. Two human IL-12 receptor proteins, designated IL-12R beta 1 and IL-12R beta 2, have been previously identified. IL-12R beta 2 has box 1 motif, box 2 motif, and three tyrosine residues in its cytoplasmic domain. In response to IL-12, Jak2 and Tyk2, family members of Janus family protein tyrosine kinases, are phosphorylated in PHA-activated T lymphocytes. The present study demonstrates that Jak2 binds to the cytoplasmic membrane-proximal region of IL-12R beta 2, and box 2 motif and tyrosine residues in the cytoplasmic domain were not required for binding. The amino-terminus of Jak2 is necessary for association with IL-12R beta 2.     

Solution assembly of the pseudo-high affinity and intermediate affinity interleukin-2 receptor complexes

The high affinity interleukin-2 receptor is composed of three cell surface subunits, IL-2Ralpha, IL-2Rbeta, and IL-2Rgamma. Functional forms of the IL-2 receptor exist, however, that enlist only two of the three subunits. On activated T-cells, the alpha- and beta-subunits combine as a preformed heterodimer (the pseudo-high affinity receptor) that serves to capture IL-2. On a subpopulation of natural killer cells, the beta- and gamma-subunits interact in a ligand-dependent manner to form the intermediate affinity receptor site. Previously, we have demonstrated the feasibility of employing coiled-coil molecular recognition for the solution assembly of a heteromeric IL-2 receptor complex. In that study, although the receptor was functional, the coiled-coil complex was a trimer rather than the desired heterodimer. We have now redesigned the hydrophobic heptad sequences of the coiled-coils to generate soluble forms of both the pseudo-high affinity and the intermediate affinity heterodimeric IL-2 receptors. The properties of these complexes were examined and their relevance to the physiological IL-2 receptor mechanism is discussed.     

Identification of a membrane protein, LAT-2, that Co-expresses with 4F2 heavy chain, an L-type amino acid transport activity with broad specificity for small and large zwitterionic amino acids.

We have identified a new human cDNA, L-amino acid transporter-2 (LAT-2), that induces a system L transport activity with 4F2hc (the heavy chain of the surface antigen 4F2, also named CD98) in oocytes. Human LAT-2 is the fourth member of the family of amino acid transporters that are subunits of 4F2hc. The amino acid transport activity induced by the co-expression of 4F2hc and LAT-2 was sodium-independent and showed broad specificity for small and large zwitterionic amino acids, as well as bulky analogs (e.g. BCH (2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid)). This transport activity was highly trans-stimulated, suggesting an exchanger mechanism of transport. Expression of tagged N-myc-LAT-2 alone in oocytes did not induce amino acid transport, and the protein had an intracellular location. Co-expression of N-myc-LAT-2 and 4F2hc gave amino acid transport induction and expression of N-myc-LAT-2 at the plasma membrane of the oocytes. These data suggest that LAT-2 is an additional member of the family of 4F2 light chain subunits, which associates with 4F2hc to express a system L transport activity with broad specificity for zwitterionic amino acids. Human LAT-2 mRNA is expressed in kidney > placenta > brain, liver > spleen, skeletal muscle, heart, small intestine, and lung. Human LAT-2 gene localizes at chromosome 14q11.2-13 (13 cR or approximately 286 kb from marker D14S1349). The high expression of LAT-2 mRNA in epithelial cells of proximal tubules, the basolateral location of 4F2hc in these cells, and the amino acid transport activity of LAT-2 suggest that this transporter contributes to the renal reabsorption of neutral amino acids in the basolateral domain of epithelial proximal tubule cells.     

Identification of the second subunit of the murine interleukin-5 receptor: interleukin-3 receptor-like protein, AIC2B is a component of the high affinity interleukin-5 receptor.

Murine interleukin-5 (IL-5) binds to its receptor with high and low affinity. It has been shown that the high affinity IL-5 receptor (IL-5-R) is composed of at least two membrane protein subunits and is responsible for IL-5-mediated signal transduction. One subunit of the high affinity IL-5-R is a 60 kDa membrane protein (p60 IL-5-R) whose cDNA was isolated using the anti-IL-5-R monoclonal antibody (mAb), H7. This subunit alone binds IL-5 with low affinity. The second subunit does not bind IL-5 by itself, and is expressed not only on IL-5-dependent cell lines but also on an IL-3-dependent cell line, FDC-P1. Expression of the p60 IL-5-R cDNA in FDC-P1 cells, which do not bind IL-5, reconstituted the high affinity IL-5-R. We have characterized the second subunit of the IL-5-R by using another anti-IL-5-R mAb, R52.120, and the anti-IL-3-R mAb, anti-Aic-2. The anti-Aic-2 mAb down-regulated binding of IL-5 to an IL-5-dependent cell line, Y16. Both R52.120 and anti-Aic-2 mAbs recognized membrane proteins of 130-140 kDa expressed on FDC-P1 and Y16 cells. The R52.120 mAb recognized both murine IL-3-R (AIC2A) and its homologue (AIC2B) expressed on L cells transfected with suitable cDNAs. The high affinity IL-5-R was reconstituted on an L cell transfectant co-expressing AIC2B and p60 IL-5-R, whereas only the low affinity IL-5-R was detected on a transfectant co-expressing AIC2A and p60 IL-5-R.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of amino acids in antiplasmin involved in its noncovalent 'lysine-binding-site'-dependent interaction with plasmin.

The lysine-binding-site-mediated interaction between plasmin and antiplasmin is of great importance for the fast rate of this reaction. It also plays an important part in regulating the fibrinolytic enzyme system. To identify structures important for its noncovalent interaction with plasmin, we constructed seven single-site mutants of antiplasmin by modifying charged amino acids in the C-terminal part of the molecule. All the variants were expressed in the Drosophila S2 cell system, purified, and shown to form stable complexes with plasmin. A kinetic evaluation revealed that two mutants of the C-terminal lysine (K452E or K452T) did not differ significantly from wild-type antiplasmin in their reactions with plasmin, in either the presence or absence of 6-aminohexanoic acid, suggesting that this C-terminal lysine is not important for this reaction. On the other hand, modification of Lys436 to Glu decreased the reaction rate about fivefold compared with wild-type. In addition, in the presence of 6-aminohexanoic acid, only a small decrease in the reaction rate was observed, suggesting that Lys436 is important for the lysine-binding-site-mediated interaction between plasmin and antiplasmin. Results from computerized molecular modelling of the C-terminal 40 amino acids support our experimental data.     

Identification of the human Mnk2 gene (MKNK2) through protein interaction with estrogen receptor beta

We have identified and characterized the human Mnk2 gene (HGMW-approved gene symbol MKNK2) through a yeast two-hybrid screen in which the Mnk2 protein interacted with the ligand-binding domain of estrogen receptor beta (ERbeta). Human Mnk2 is homologous to murine Mnk2 ( approximately 94% identical) and human Mnk1 (71% identical), both of which encode MAP kinase interacting kinases that are phosphorylated and activated by ERK1 and 2. This report presents a thorough genomic sequence analysis revealing that the human Mnk2 gene has two C-terminal splice variants, designated here as Mnk2a and Mnk2b. These two isoforms are identical over the first 385 amino acids of the coding sequence and differ only in the final exon which encodes an additional 80 residues for Mnk2a and 29 residues for Mnk2b. A more detailed biological analysis in yeast showed that the Mnk2 interaction was selective for ERbeta as opposed to ERalpha and that the interaction was specific to Mnk2b as opposed to Mnk2a or Mnk1. This pattern was reproduced in a mammalian two-hybrid system using a completely different set of fusion partners; and in both yeast and mammalian systems, the addition of estradiol decreased the interaction. While it remains unknown whether ERbeta is a substrate of Mnk2, the interaction of these two proteins is reminiscent of ERalpha and ribosomal S6 kinase (p90-RSK), another MAP kinase-regulated kinase homologous to Mnk2 that is known to phosphorylate ERalpha.     

The third molecule associated with interleukin 2 receptor alpha and beta chain.

It is known that the affinity cross-linking study of the human high-affinity Interleukin 2 (IL-2) receptor reveals triplet bands consisting of 70 kDa alpha chain(Tac)-IL-2 and the 90/80 kDa doublet. We found the cell lines lacking the lower band of the doublet in spite of the expression of both alpha and beta chains. No IL-2 binding was detectable in the presence of anti-Tac antibody in these cells. Immunoprecipitation from the cell extract of [125 I] IL-2-cross-linked T cells with anti-beta chain polyclonal IgG detected the upper band, but not lower band of the doublet. These data suggest that the lower band of the doublet represents an unknown IL-2-binding protein (p65) distinct from the beta chain and this molecule may be involved in the intermediate-affinity IL-2 binding together with the beta chain.     

Biochemical analysis of interleukin-2 receptor beta chain phosphorylation by p56(lck)

Tyrosine phosphorylation of multiple proteins, including the receptor itself, is an initial event in IL-2 signaling and leads to recruitment of SH2 or PTB domain-containing proteins to the receptor. In this study, we have used subdomains of the IL-2 receptor beta chain (IL-2Rbeta) expressed in Escherichia coli as GST fusion proteins to identify the tyrosine residues that could be phosphorylated by p56(lck), one of the critical tyrosine kinases activated by IL-2. We report that recombinant p56(lck) phosphorylates in vitro tyrosine residues within the IL-2Rbeta chain but not those within the IL-2Rgamma chain. p56(lck) phosphorylates tyrosine residues 355, 358 and 361 but not 338 of the IL-2Rbeta chain acidic subdomain. Interestingly, phosphorylation of Tyr-358 appears to require the presence of either Tyr-355 or Tyr-361. p56(lck) also phosphorylates very efficiently the two tyrosines present in the IL-2Rbeta chain C-terminal region, Tyr-392 and Tyr-510. We also investigated the association of p56(lck) with the IL-2Rbeta chain which was found to depend on a short stretch of the IL-2Rbeta chain acidic subdomain, and to be independent of the presence of its tyrosine residues.     

Expression and characterization of a des-methionine mutant interleukin-2 receptor (Tac protein) with interleukin-2 binding affinity

A gene coding for the Tac protein (interleukin-2 receptor alpha-subunit, IL-2R alpha) of the interleukin-2 receptor was constructed by chemoenzymatic gene synthesis. The gene designed for mutagenesis codes for a receptor protein where all 10 methionines are substituted by alanine, valine, leucine, and isoleucine. In addition, aspartate at position 6 is substituted by glutamate. This desmethionine IL-2R alpha and the wild-type IL-2R alpha genes were integrated into a eukaryotic expression vector and transferred into different cell lines. The recipient cell lines express both wild-type and mutant receptor proteins on their cell surfaces which are recognized equally by different monoclonal antibodies. It was possible to establish cell lines with high level IL-2R alpha chain expression by fluorescence-activated cell sorting. The wild-type IL-2R alpha expressed in LTK- cells is a glycoprotein with an apparent molecular size of about 60 kDa and a typical low interleukin-2 binding affinity of KD = 12 nM. Despite the fact that 11 amino acids are altered, no significant difference in the mutant IL-2R alpha is observed, exhibiting the same molecular size and a low interleukin-2 binding affinity of KD = 26 nM.