Cardiac myosin motor deficits are associated with left ventricular dysfunction in human ischemic heart failure.

During ischemic heart failure (IHF), cardiac muscle contraction is typically impaired, though the molecular changes within the myocardium are not fully understood. Thus, we aimed to characterize the biophysical properties of cardiac myosin in IHF. Cardiac tissue was harvested from 10 age-matched males, either with a history of IHF or nonfailing (NF) controls that had no history of structural or functional cardiac abnormalities. Clinical measures before cardiac biopsy demonstrated significant differences in measures of ejection fraction and left ventricular dimensions. Myofibrils and myosin were extracted from left ventricular free wall cardiac samples. There were no changes in myofibrillar ATPase activity or calcium sensitivity between groups. Using isolated myosin, we found a 15% reduction in the IHF group in actin sliding velocity in the in vitro motility assay, which was observed in the absence of a myosin isoform shift. Oxidative damage (carbonylation) of isolated myosin was compared, in which there were no significant differences between groups. Synthetic thick filaments were formed from purified myosin and the ATPase activity was similar in both basal and actin-activated conditions (20 µM actin). Correlation analysis and Deming linear regression were performed between all studied parameters, in which we found statistically significant correlations between clinical measures of contractility with molecular measures of sliding velocity and ELC carbonylation. Our data indicate that subtle deficits in myosin mechanochemical properties are associated with reduced contractile function and pathological remodeling of the heart, suggesting that the myosin motor may be an effective pharmacological intervention in ischemia.NEW & NOTEWORTHY Ischemic heart failure is associated with impairments in contractile performance of the heart. This study revealed that cardiac myosin isolated from patients with ischemic heart failure had reduced mechanical activity, which correlated with the impaired clinical phenotype of the patients. The results suggest that restoring myosin function with pharmacological intervention may be a viable method for therapeutic intervention.

KIR2DL4-HLAG interaction at human NK cell-oligodendrocyte interfaces regulates IFN-γ-mediated effects.

Interactions between germline-encoded natural killer (NK) cell receptors and their respective ligands on tumorigenic or virus-infected cells determine NK cell cytotoxic activity and/or cytokine secretion. NK cell cytokine responses can be augmented in and can potentially contribute to multiple sclerosis (MS), an inflammatory disease of the central nervous system focused upon the oligodendrocytes (OLs). To investigate mechanisms by which NK cells may contribute to MS pathogenesis, we developed an in vitro human model of OL-NK cell interaction. We found that activated, but not resting human NK cells form conjugates with, and mediate cytotoxicity against, human oligodendrocytes. NK cells, when in conjugate with OLs, rapidly synthesize and polarize IFN-γ toward the OLs. IFN-γ is capable of reducing myelin oligodendrocyte and myelin associated glycoproteins (MOG and MAG) content. This activity is independent of MHC class-I mediated inhibition via KIR2DL1, but dependent upon the interaction between NK cell-expressed KIR2DL4 and its oligodendrocyte-expressed ligand, HLA-G. NK cells from patients with MS express higher levels of IFN-γ following conjugation to OLs, more actively promote in vitro reduction of MOG and MAG and have higher frequencies of the KIR2DL4 positive population. These data collectively suggest a mechanism by which NK cells can promote pathogenic effects upon OLs.

Human natural killer cell receptors and signal transduction.

Natural killer (NK) cells express numerous receptors, which continually engage with ligands on cell surfaces. Until 1995, only a handful of these receptors were characterized and the molecular basis of NK cell activation was obscure. Recently, considerable advances have been made in characterizing the receptor repertoire on human NK cells. Both activating and inhibitory receptors can transduce positive or negative signals to regulate NK cell cytotoxicity and cytokine release responses. The inhibitory receptors normally predominate in this balance of signals. Certain tumor cells and virally infected cells that lack major histocompatibility complex (MHC) class I molecules, however, can rapidly trigger NK cell activation. The basis of this activation is the loss of negative signals that are normally transmitted by MHC class I-binding inhibitory receptors, and the corresponding domination of activating receptor signals. While ligand specificity for a number of the recently described receptors is still a mystery, their signal transduction properties have begun to be defined. The dynamic crosstalk between these receptors ultimately governs the NK cell activation state. Although the complexities of NK cell signalling are only marginally understood, several overall themes have been defined by characterizing the roles of distinct pathways during NK cell responses.

DAP12: a key accessory protein for relaying signals by natural killer cell receptors.

DAP12 is a 12 kDa transmembrane protein recently recognized as a key signal transduction receptor element in Natural Killer (NK) cells. It is a disulfide-linked homodimer that non-covalently associates with several activating receptors expressed on NK cells. Activation signals initiated through DAP12 are predicted to play strategic roles in triggering NK cell cytotoxicity responses toward certain tumor cells and virally infected cells. The cytoplasmic domain of DAP12 contains an Immunoreceptor Tyrosine-based Activation Motif (ITAM). Phosphorylation of ITAM tyrosines mediates associations with protein tyrosine kinases, which is a resonant feature of signalling through these motifs in T and B cell antigen receptors. In addition, its expression in other tissues, including dendritic cells and monocytes, suggests that DAP12 transduces ITAM-mediated activation signals for an extended array of receptors in those cells as well.

Interaction of p59fyn kinase with the dynein light chain, Tctex-1, and colocalization during cytokinesis.

The protein tyrosine kinase p59fyn (Fyn) plays important roles in both lymphocyte Ag receptor signaling and cytokinesis of proB cells. We utilized yeast two-hybrid cloning to identify the product of the tctex-1 gene as a protein that specifically interacts with Fyn, but not with other Src family kinases. Tctex-1 was recently identified as a component of the dynein cytoskeletal motor complex. The capacity of a Tctex-1-glutathione S-transferase fusion protein to effectively bind Fyn from cell lysates confirmed the authenticity of this interaction. Tctex-1 binding required the first 19 amino acids of Fyn and integrity of two lysine residues within this sequence that were previously shown to be important for Fyn interactions with the immunoreceptor tyrosine-based activation motifs (ITAMs) of lymphocyte Ag receptors. Expression of tctex-1 mRNA and protein was observed in all lymphoma lines analyzed, and immunofluorescence confocal microscopy localized the protein to the perinuclear region. Analysis of a T cell hybridoma revealed prominent colocalization of Tctex-1 and Fyn at the cleavage furrow and mitotic spindles in cells undergoing cytokinesis. Our results provide a unique insight into a mechanism by which Tctex-1 might mediate specific recruitment of Fyn to the dynein complex in lymphocytes, which may be a critical event in mediating the previously defined role of Fyn in cytokinesis.

The Ig alpha/Igbeta heterodimer on mu-negative proB cells is competent for transducing signals to induce early B cell differentiation.

The immunoglobulin alpha (Ig alpha)/Ig beta heterodimer was detected on the surface of mu-negative proB cell lines in association with calnexin. The cross-linking of Ig beta on proB cells freshly isolated from bone marrow of recombination activating gene (RAG)-2-deficient mice induced a rapid and transient tyrosine-phosphorylation of Ig alpha as well as an array of intracellular proteins including Syk, PI3-kinase, Vav, and SLP-76. It also elicited the phosphorylation and activation of a MAP kinase ERK but not JNK/SAPK or p38. When RAG-2-deficient mice were treated with anti-Ig beta monoclonal antibody, developmentally arrested proB cells were induced to differentiate to the small preB cell stage as observed when the mu transgene was expressed in RAG-2-deficient mice. Thus, the cross-linking of Ig beta on proB cells appears to elicit differentiation signals analogous to those delivered by the preB cell receptor in normal B cell development.

Inactivation of pRB-related proteins p130 and p107 mediated by the J domain of simian virus 40 large T antigen.

Inactivation of the retinoblastoma tumor suppressor protein (pRB) contributes to tumorigenesis in a wide variety of cancers. In contrast, the role of the two pRB-related proteins, p130 and p107, in oncogenic transformation is unclear. The LXCXE domain of simian virus 40 large T antigen (TAg) specifically binds to pRB, p107, and p130. We have previously shown that the N terminus and the LXCXE domain of TAg cooperate to alter the phosphorylation state of p130 and p107. Here, we demonstrate that TAg promotes the degradation of p130 and that the N terminus of TAg is required for this activity. The N terminus of TAg has homology to the J domain of the DnaJ family of molecular chaperone proteins. Mutants with mutations in the J-domain homology region of TAg are defective for altering p130 and p107 phosphorylation and for p130 degradation. A heterologous J-domain from a human DnaJ protein can functionally substitute for the N terminus of TAg in the effect on p107 and p130 phosphorylation and p130 stability. We further demonstrate that the J-domain homology region of TAg confers a growth advantage to wild-type mouse embryo fibroblasts (MEFs) but is dispensable in the case of MEFs lacking both p130 and p107. This indicates that p107 and p130 have overlapping growth-suppressing activities whose inactivation is mediated by the J domain of TAg.

DnaJ/hsp40 chaperone domain of SV40 large T antigen promotes efficient viral DNA replication.

The amino-terminal domain of SV40 large tumor antigen (TAg) is required for efficient viral DNA replication. However, the biochemical activity associated with this domain has remained obscure. We show here that the amino-terminal domain of TAg shares functional homology with the J-domain of DnaJ/hsp40 molecular chaperones. DnaJ proteins function as cofactors by regulating the activity of a member of the 70-kD heat shock protein family. Genetic analyses demonstrated that amino-terminal sequences of TAg comprise a novel J-domain that mediates a specific interaction with the constitutively expressed hsc70 and show that the J-domain is also required for efficient viral DNA replication in vivo. Furthermore, we demonstrated that the J-domain of two human DnaJ homologs, HSJ1 or DNAJ2, could substitute functionally for the amino-terminus of TAg in promoting viral DNA replication. Together, our findings suggest that TAg uses its J-domain to support SV40 DNA replication in a manner that is strikingly similar to the use of Escherichia coli DnaJ by bacteriophage lambda in DNA replication. However, TAg has evolved a more efficient strategy of DNA replication through an intrinsic J-domain to associate directly with a partner chaperone protein. Our observations provide evidence of a role for chaperone proteins in the process of eukaryotic DNA replication.

The cytoplasmic domain of rat NKR-P1 receptor interacts with the N-terminal domain of p56(lck) via cysteine residues.

NKR-P1 is a type II transmembrane protein which acts as an activation receptor on natural killer (NK) cells. The cytoplasmic domains of the CD4, CD8 and 4-1BB receptors contain the sequence Cys-X-Cys-Pro which is directly involved in coupling to another pair of cysteines in the N-terminal domain of the src family tyrosine kinase p56(lck). The cytoplasmic domain of NKR-P1 in rodents also contains the Cys-X-Cys-Pro sequence, but the capacity of the receptor to bind p56(lck) is presently unknown. We tested for direct coupling between these proteins using both protein biochemistry and the yeast two-hybrid technique. Immunoprecipitation studies showed that p56(lck) can be co-immunoprecipitated with NKR-P1 from a rat NK tumor cell line. In addition, the cytoplasmic domain of NKR-P1 interacted with the N-terminal domain of p56(lck) in yeast as assessed by reporter gene activation. Integrity of the cysteine pairs in both proteins was critical in mediating the interaction. The experiments suggest that the association of p56(lck) with NKR-P1 is somewhat weaker than the p56(lck) association with CD8alpha, but of much lower avidity than between CD4 and p56(lck). This could reflect a higher activation threshold for the NKR-P1 and CD8 receptors, which are involved in cytolytic responses, compared to CD4 which is involved in T cell helper function.

Tyrosine phosphorylation of a human killer inhibitory receptor recruits protein tyrosine phosphatase 1C.

Natural killer (NK) cells express killer inhibitory receptors that mediate negative regulation of NK cell cytotoxicity upon binding to MHC class I molecules on target cells. Unrelated inhibitory receptors on B cells have recently been shown to function through recruitment of phosphotyrosine phosphatase 1C (PTP-1C). Here, we show that a human killer inhibitory receptor specific for HLA-C also recruits PTP-1C after phosphorylation induced either by the pharmacological agent phenylarsine oxide or by conjugation with target cells. This recruitment is mediated by the binding of specific cytoplasmic phosphotyrosine-containing sequences to PTP-1C. These results implicate PTP-1C as a cytosolic component of the negative signaling pathway through NK cell inhibitory receptors.

Manipulation of B cell antigen receptor tyrosine phosphorylation using aluminum fluoride and sodium orthovanadate.

The B cell antigen receptor complex (BCR) is composed of a membrane-spanning immunoglobulin molecule (mIg) non-covalently associated with heterodimers of the transmembrane proteins Ig-alpha and Ig-beta. The cytoplasmic domains of Ig-alpha and Ig-beta do not contain kinase domains but are phosphorylated on tyrosine residues immediately upon receptor ligation. The mechanism and kinase responsible for initial Ig-alpha and Ig-beta phosphorylation following receptor ligation is unknown, In an attempt to better understand this process, Ig-alpha and Ig-beta phosphorylation was examined in response to treatment of permeabilized B cells with the pharmacologic agents, aluminum fluoride (AlFx) and sodium orthovanadate (Na3VO4). AlFx is known to stimulate GTP-binding proteins while Na3VO4 inhibits protein tyrosine phosphatases (PTPs), both of which are involved in the BCR signalling cascade. In these studies, AlFx and Na3VO4 stimulated rapid tyrosine phosphorylation of Ig-alpha, Ig-beta, and additional cellular proteins, including the protein tyrosine kinase (PTK) Lyn. The tyrosine phosphorylation does not appear to be mediated through GTP-binding proteins, since GTP gamma S did not stimulate tyrosine phosphorylation. As expected, however, PTPs modulate the phosphorylation state of these proteins since another PTP inhibitor, phenylarsine oxide (PAO), increased phosphorylation of Ig-alpha, Ig-beta and other proteins in this system. Interestingly, the extent and kinetics of the mIg-associated Lyn and Ig-alpha/Ig-beta phosphorylation was correlated, suggesting that Lyn may mediate receptor phosphorylation. Alternatively, Lyn, may be a downstream effector of phosphorylated Ig-alpha and Ig-beta as suggested by the reported ability of biphosphorylated Ig-alpha to activate Fyn PTK in vitro. Finally, all components necessary for Na3VO4, but not AlFx, stimulation of phosphorylation are membrane associated. The data are consistent with modulation of phosphorylation of Ig-alpha and Ig-beta through both PTP inhibition and AlFx treatment, and a common intermediary in or effector of these phosphorylation pathways appears to be the Lyn kinase.

Interactions between the amino-terminal domain of p56lck and cytoplasmic domains of CD4 and CD8 alpha in yeast.

The interactions between CD4 or CD8 and p56lck were tested using the two-hybrid protein interaction system in yeast. Plasmid constructs were created which fuse the cytoplasmic domains of either CD4 or CD8 alpha to the DNA-binding protein LexA, and the unique amino-terminal domain of p56lck fused to a transcriptional activation domain. These constructs were transfected into yeast bearing lacZ and LEU2 reporter genes controlled by upstream LexA operator sequences. Yeast transfectants bearing either CD4 or CD8 alpha hybrid proteins in combination with the amino terminal p56lck hybrid protein exhibited increased beta-galactosidase activity and growth on leucine-deficient medium, indicating interactions between these protein domains. Quantitation of reporter activation indicated that the interaction of p56lck with CD8 alpha is at least 18-fold weaker than the interaction with CD4 in this assay. This reduced interactive capacity is apparently not due to competition by CD8 alpha interacting with itself, since homotypic or heterotypic interactions between CD8 alpha and/or CD4 could not be detected. Truncation and point mutants demonstrated that the interactions of p56lck with CD4 or CD8 alpha were dependent on the integrity of a pair of cysteines on each protein. The results indicate that these interactions do not require any additional proteins. Additionally, expression of the entire p56lck molecule as a hybrid with LexA resulted in dramatic reduction in the growth of yeast. Though the two-hybrid system is a powerful tool for examining protein interactions, this result indicates potential limitations in studying full-length src family tyrosine kinases in yeast.

Identification of regions in polyomavirus middle T and small t antigens important for association with protein phosphatase 2A.

Two subunits of protein phosphatase 2A (PP2A) have been shown previously to bind to the small t and middle T antigens (ST and MT, respectively) of polyomavirus. To determine sequences important for binding of PP2A to ST and MT, we first constructed a series of ST mutants in regions known to be important for biological activity of ST and MT. Several mutations in two small regions just amino terminal to the Cys-X-Cys-X-X-Cys motifs of ST and MT abolished PP2A binding to ST in vitro. Parallel mutations were constructed in MT to investigate the role of PP2A binding in the function of polyomavirus MT. Wild-type and mutant MT proteins were stably expressed in NIH 3T3 cells and analyzed (i) for their ability to induce transformation and (ii) for associated cellular proteins and corresponding enzymatic activities previously described as associating with wild-type MT. A number of the mutant MTs were found to be defective in binding of PP2A as assayed by coimmunoprecipitation. In contrast, a deletion of the highly conserved stretch of amino acids 42 to 47 (His-Pro-Asp-Lys-Gly-Gly) in the ST-MT-large T antigen common region did not affect PP2A binding to MT. MT mutants defective for PP2A binding were also defective in transformation, providing further evidence that association with PP2A is important for the ability of MT to transform cells. All mutants which were impaired for PP2A binding were similarly or more dramatically impaired for associated protein and lipid kinase activities, supporting the possibility that PP2A binding is necessary for the formation and/or stability of an MT-pp60c-src complex.

CART: a conserved antigen receptor transmembrane motif.

We have compared the transmembrane sequences of 72 vertebrate antigen receptor (mIg and TCR) polypeptides. This allowed us to identify a Conserved Antigen Receptor Transmembrane (CART) motif which is present in all antigen receptor transmembrane domains from species as far removed as cartilaginous fish. Most of the amino acids in the CART motif are polar or aromatic and may interact with other proteins in the lipid environment. In addition, modeling the antigen receptor transmembrane domain in an alpha helical conformation places the CART residues on one face of the alpha helix. Thus, the CART motif may encode a structural unit which plays a role in the assembly and/or the signaling properties of lymphocyte antigen receptors. We speculate on the potential role of the CART motifs in lymphocyte signaling.

Polyoma middle tumor antigen interacts with SHC protein via the NPTY (Asn-Pro-Thr-Tyr) motif in middle tumor antigen.

Polyomavirus middle tumor antigen (MT) transforms a large number of cell types by binding to and modulating the activities of cellular proteins. Previous genetic analysis defined in MT an independent motif, NPTY (Asn-Pro-Thr-Tyr), required for transformation. This report demonstrates that NPTY is required for interaction between MT and SHC protein, a Src homology 2 (SH2)-containing protooncogene product implicated in activating Ras via association with GRB2 protein. SHC is phosphorylated on tyrosine and associates with GRB2 in MT-transformed cells. These effects require an intact NPTY motif in MT. SHC immunoprecipitates from MT-transformed cells possess kinase activity that phosphorylates not only SHC and MT but also the 85-kDa subunit of phosphatidylinositol 3-kinase. This result suggests that a complex exists that contains, at a minimum, MT, Src family tyrosine kinases, phosphatidylinositol 3-kinase, and SHC.

CDw50 and ICAM-3: two names for the same molecule.

CDw50 differentiation antigen is a molecule broadly expressed on hematopoetic cells but not on other cells. Previous experiments showed that CDw50 monoclonal antibodies (mAb) inhibited primary mixed lymphocyte culture (MLC). To understand the function of CDw50 better, we purified it and obtained peptide sequence. At the same time, intercellular adhesion molecule (ICAM)-3, the third ligand of lymphocyte function-associated molecule 1, was described by mAb and subsequent cDNA cloning. Immunochemical, functional, and protein sequencing studies show that ICAM-3 and CDw50 are the same glycoprotein, a 120-kDa surface molecule with presumably an important role in the immune responses.

The gamma subunit of the B cell antigen-receptor complex is a C-terminally truncated product of the B29 gene.

The predominant Ag-receptor complex of B cells consists of mIgM or mIgD noncovalently associated with glycosylated heterodimers of Ig-alpha and Ig-beta or Ig-alpha and Ig-gamma. Upon B cell stimulation the associated proteins are phosphorylated, giving rise to pp32/33 (alpha), pp37 (beta), and pp34 (previously designated gamma). Ig-alpha and Ig-beta contain extended cytoplasmic structure (61 and 48 amino acids, respectively) and associate with cytoplasmic effectors indicating that they are directly involved in signal transduction. Here we report analysis of the structural relationship of mIgM- and mIgD-associated Ig-beta and Ig-gamma chains from mice. N-terminal sequence, immunoblotting, and physicochemical analyses show that both Ig-beta and Ig-gamma are products of the B cell-specific B29 gene and demonstrate that the 37-kDa Ig-beta protein is the full length predicted product of the B29 gene. The Ig-associated protein that migrates in the 34-kDa range is actually two distinct species. The minor species is a phosphorylatable and underglycosylated form of full length Ig-beta, and the major species is a C-terminally truncated form of B29, which we now designate Ig-gamma. This conclusion is based on the observations that Ig-gamma is composed of a core protein which is 3 to 4 kDa smaller than deglycosylated Ig-beta, it is not phosphorylated, unlike Ig-beta, and it does not react with an antiserum raised against a peptide of the seven C-terminal amino acids of B29. Based on these findings we estimate that Ig-gamma is truncated by about 30 to 36 amino acid residues and hypothesize that the most 3' B29 exon, which encodes the 32 C-terminal residues, may not be expressed in Ig-gamma. All of the documented B29 products are found in association with both mIgM and mIgD. Interestingly, Ig-gamma is found in intermediate and low density splenic B cells, but is not detectable in resting B cells. This raises the possibility that it may confer some distinct signaling function on the Ag receptors of these cells.