Association of the 72-kDa protein-tyrosine kinase PTK72 with the B cell antigen receptor.

The antigen receptors on B lymphocytes are cell-surface immunoglobulins. Antibodies against surface IgM (sIgM) coimmunoprecipitate several sIgM-associated proteins. Incubation of anti-IgM complexes with [gamma-32P]ATP leads to the phosphorylation on tyrosine of IgM-associated proteins including MB-1 and a protein of 72 kDa. Peptide mapping and reimmunoprecipitation experiments indicate that the 72-kDa phosphoprotein is PTK72, a protein-tyrosine kinase that is expressed at highest levels in B lymphocytes. MB-1 is also phosphorylated in immune complexes prepared with antibodies to PTK72, indicating that components of the IgM complex are associated with PTK72. In addition, PTK72 is associated with sIgD complexes isolated from spleen B lymphocytes. The cross-linking of sIgM antigen receptors on B lymphocytes leads to the rapid phosphorylation of PTK72 on tyrosine and to the activation of PTK72 as measured by autophosphorylation and by the phosphorylation of an exogenous substrate in anti-PTK72 immune complexes. These results suggest that the signaling cascade initiated by engagement of the B cell antigen receptor involves the increased enzymatic activity of PTK72, which is already present in a preformed antigen receptor complex.     

Synthesis and conformation of the trimeric coiled-coil segment of laminin.

A disulfide linked 95-mer parallel hetero-trimeric active site segment of laminin was designed and synthesised. The three subunits, A (32-mer), B1 (30-mer) and B2 (33-mer), were prepared by Boc-based solid-phase peptide synthesis involving a two-step trimethylsilyl bromide-thioanisole and HF deprotection procedure. The interlinking of the three subunits was accomplished by the stepwise selective formation of two disulfide bridges using air-oxidation and thallium (III) trifluoroacetate oxidation. The conformations of the synthetic peptides were studied by circular dichroism (CD) spectroscopy, showing that the hetero-dimer, B1-B2, one of the homo-dimers, B1-B1, and the trimer are 30 to 40% in the alpha-helical conformation in aqueous buffer. Variable temperature CD studies demonstrated that the trimer is considerably more stable (melting temperature (Tm) = 61 degrees) than the hetero-dimer, B1-B2 (Tm = 36 degrees).     

Dopamine and cyclic AMP-regulated phosphoprotein-32 phosphorylation pattern in cocaine and morphine-sensitized rats

This study reports some of the modifications in dopaminergic signalling that accompany cocaine and morphine behavioural sensitization. Cocaine-sensitized rats showed increased phosphorylation of dopamine- and cyclic AMP-regulated phosphoprotein Mr 32 kDa (DARPP-32) at threonine-75 (Thr75) and decreased DARPP-32 phosphorylation at Thr34, in the caudate-putamen (CPu) and nucleus accumbens (NAc) 7 days after sensitization assessment. Conversely, in morphine-sensitized rats, no apparent modifications in DARPP-32 phosphorylation pattern were observed. Morphine-sensitized rats have increased binding and coupling of micro -opioid receptors and increased dopaminergic transmission in striatal areas and, upon morphine challenge, exhibit dopamine D1 receptor-dependent stereotypies. Thus, the DARPP-32 phosphorylation pattern was studied in morphine-sensitized rats at different times after morphine challenge. Morphine challenge increased levels of phospho-Thr75 DARPP-32 and decreased levels of phospho-Thr34 DARPP-32 in a time-dependent manner in the CPu and NAc. In order to assess whether these modifications were related to modified cyclic AMP-dependent protein kinase (PKA) activity, the phosphorylation levels of two other PKA substrates were examined, the GluR1 and NR1 subunits of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate and NMDA receptors respectively. The phosphorylation levels of GluR1 and NR1 subunits decreased in parallel with those of phospho-Thr-34 DARPP-32, supporting the hypothesis that morphine challenge elicited a decrease in PKA activity in morphine-sensitized rats.     

Membrane immunoglobulin: anti-immunoglobulin interactions mediate the phosphorylation of actin associated proteins in the B-lymphocyte

The binding of membrane immunoglobulin (mIg) by anti-Ig antibodies is known to initiate a mitogenic signal in B lymphocytes. Because in many instances growth control appears to be correlated with phosphokinase activity, as well as with alterations in cytoskeletal architecture, we asked the question whether antibodies binding to mIg would also lead to the specific phosphorylation of lymphocyte actin-associated proteins. Utilizing a myosin affinity technique, we directly examined phosphoproteins that were associated with actin in the chicken B cell. We found that in a few instances the level of phosphorylation was indeed modulated by mIg:anti-Ig interactions. These actin-binding phosphoproteins may be important control elements in the lymphocyte cytoskeleton.     

Regulation of DARPP-32 dephosphorylation at PKA- and Cdk5-sites by NMDA and AMPA receptors: distinct roles of calcineurin and protein phosphatase-2A

Glutamatergic inputs from corticostriatal and thalamostriatal pathways have been shown to modulate dopaminergic signaling in neostriatal neurons. DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of M (r) 32 kDa) is a signal transduction molecule that regulates the efficacy of dopamine signaling in neostriatal neurons. Dopamine signaling is mediated in part through phosphorylation of DARPP-32 at Thr34 by cAMP-dependent protein kinase, and antagonized by phosphorylation of DARPP-32 at Thr75 by cyclin-dependent protein kinase 5. We have now investigated the effects of the ionotropic glutamate NMDA and AMPA receptors on DARPP-32 phosphorylation in neostriatal slices. Activation of NMDA and AMPA receptors decreased the state of phosphorylation of DARPP-32 at Thr34 and Thr75. The decrease in Thr34 phosphorylation was mediated through Ca(2+) -dependent activation of the Ca(2+) -/calmodulin-dependent phosphatase, calcineurin. In contrast, the decrease in Thr75 phosphorylation was mediated through Ca(2+) -dependent activation of dephosphorylation by protein phosphatase-2A. The results provide support for a complex effect of glutamate on dopaminergic signaling through the regulation of dephosphorylation of different sites of DARPP-32 by different protein phosphatases.     

Glucocorticoid receptor phosphorylation in mouse L-cells

This paper summarizes our observations on the phosphorylation state of untransformed and transformed glucocorticoid receptors isolated from 32P-labeled L-cells. The 300-350-kDa 9S untransformed murine glucocorticoid receptor complex is composed of a 100-kDa steroid-binding phosphoprotein and one or possibly two units of the 90-kDa heat shock protein (hsp90), which is also a phosphoprotein. Transformation of this complex to the 4S DNA-binding state is accompanied by dissociation of hsp90. When receptors in cytosol are transformed by heating at 25 degrees C, there is no gross change in the degree of phosphorylation of the steroid-binding protein. Both receptors that are bound to DNA after transformation under cell-free conditions and receptors that are located in the nucleus of cells incubated at 37 degrees C in the presence of glucocorticoid are labeled with 32P. The results of experiments in which the 32P-labeled receptor was submitted to limited proteolysis suggest that the 16-kDa DNA-binding domain is phosphorylated and that the 28-kDa steroid-binding domain is not.     

Membrane Ig-cytoskeletal interactions. I. Flow cytofluorometric and biochemical analysis of membrane IgM-cytoskeletal interactions

Membrane IgM (mIgM) and mIgD are the receptors for Ag on the surface of B lymphocytes, mIg is soluble in detergent; however, when mIg is cross-linked with anti-Ig, the mIg becomes associated with the cytoskeletal matrix and is rendered detergent-insoluble. By a novel flow cytofluorometric assay and by biochemical analysis, it has been shown that anti-isotype-specific antibodies induce mIgM and mIgD to associate with the cytoskeleton of B lymphocytes in an isotype-specific fashion. The detergent solubility of other prominent B lymphocyte surface proteins, such as class I and class II MHC proteins were unaffected by cross-linking of mIg. A panel of mu-specific mAb was analyzed for their ability to induce mIgM-cytoskeletal association. Although all mAb bound mIgM, only three out of seven rendered mIgM cytoskeletally associated. Further analysis revealed a strict correlation in the capacity of mu-specific mAb to induce capping and to induce the association of mIgM with the cytoskeleton.     

T helper cell-dependent B cell activation.

Small, resting B lymphocytes are driven into the cell cycle as a consequence of receiving multiple signals from elements found within their local environment. The first of these signals results from the binding of specific antigen to membrane immunoglobulin (mIg) receptors on the B cells. Pursuant to antigen binding, signals are transduced and the B cell commences to endocytose and degrade the antigen. Fragments of the antigen are expressed on the B cell surface in noncovalent association with class II major histocompatibility complex (MHC) molecules. The antigen-class II MHC complex serves as a recognition complex for CD4+ helper T cells (Th). As a consequence of recognition, Th form stable physical conjugates with the B cells. Over an extended period of time the Th and B cells bilaterally signal one another. This interchange of signals results in the growth and differentiation of both cells. This review will discuss the sequence of events that culminate in the growth and differentiation of B lymphocytes to antibody-producing cells.     

Insulin and insulin-like growth factor-I stimulate a common endogenous phosphoprotein substrate (pp185) in intact neuroblastoma cells

Mouse neuroblastoma N18 cells contain specific high affinity insulin and insulin-like growth factor-I (IGF-I) receptors. Insulin and IGF-I induce phosphorylation, in intact cells, of their respective receptor beta subunits. The insulin receptor beta subunit is represented by a 95-kDa phosphoprotein that is recognized by a specific antiserum (B10). The IGF-I receptor beta subunit is represented by two phosphoproteins of molecular mass 95 and 105 kDa. The hormone-induced phosphorylation was rapid and dose-dependent occurring on both phosphoserine and phosphotyrosine residues. In addition, both insulin and IGF-I induced phosphorylation of an endogenous protein of molecular mass 185 kDa (pp185). The rapidity and dose dependency of the phosphorylation of pp185 suggested that it may represent a common endogenous substrate for the insulin and IGF-I receptors in these neural-derived cells. Phosphorylation was primarily on phosphoserine and phosphotyrosine residues. pp185 did not absorb to wheat germ agglutinin-agarose and was not stimulated by either epidermal growth factor or platelet-derived growth factor. The finding of pp185 in these neural-related cells as well as in non-neural tissues suggests that it may represent a ubiquitous endogenous substrate for both the insulin and IGF-I receptor kinases.     

Vav-dependent and vav-independent phosphatidylinositol 3-kinase activation in murine B cells determined by the nature of the stimulus

We show in this study that B cell activation following high avidity ligation of IgM or coligation of membrane Ig with CD19 elicits similar levels of Ca(2+) flux using different mechanisms. Each form of activation requires the function of Vav and PI3K. However, Vav regulates Ca(2+) flux independently of PI3K following anti-IgM cross-linking. By contrast, Vav function is essential for PI3K activation following membrane Ig (mIg)/CD19 coligation. Inhibition of PI3K revealed anti-IgM-stimulated Ca(2+) flux has a PI3K-independent component, while Ca(2+) flux following mIg/CD19 coligation is totally PI3K dependent. The p85alpha and p110delta subunits of PI3K both participate in anti-IgM and mIg/CD19 coligation-induced Ca(2+) flux, although the defects are not as severe as observed after pharmacological inhibition. This may reflect the recruitment of additional PI3K subunits, as we found that p110alpha becomes associated with CD19 upon B cell activation. These data show that the nature of the Ag encountered by B cells determines the contribution of Vav proteins to PI3K activation. Our results indicate that the strong signals delivered by multivalent cross-linking agents activate B cells in a qualitatively different manner from those triggered by coreceptor recruitment.     

Temperature-mediated processes in teleost immunity: the effects of temperature on membrane immunoglobulin capping on channel catfish B lymphocytes

1. In order to better understand ligand-induced redistribution of membrane receptors and lymphocyte activation in ectothermic vertebrates, flow cytometry was used to monitor the effects of both in vivo acclimation temperature and in vitro assay temperatures on the kinetics of monoclonal antibody-induced membrane immunoglobulin (mIg) capping on channel catfish lymphocytes. 2. It was observed that the kinetics of mIg capping were dependent on in vitro assay temperatures, in vivo acclimation temperatures, and the length of time of in vivo acclimation. In the latter situation in vivo acclimation of fish to 27, 22 and 17 degrees C was considered complete after 3 weeks, while acclimation to 12 degrees C required a minimum of 5 weeks. 3. The energies of activation required for mIg capping ranged from 33 to 24 kcal/mol; lower energies of activation were observed with lower temperature acclimation. 4. It was also noted that the lower energies of activation were associated with concomitant decreases in cellular phospholipid saturated/unsaturated fatty acid ratios. 5. It appears that channel catfish B cell mIg capping, presumably a requisite for immune function, can be significantly affected by environmental temperatures; most likely such effects are attributable to changes in plasma membrane viscosities.     

Lymphocyte lineage-restricted tyrosine-phosphorylated proteins that bind PLC gamma 1 SH2 domains.

Epidermal growth factor (EGF) or platelet-derived growth factor binding to their receptor on fibroblasts induces tyrosine phosphorylation of PLC gamma 1 and stable association of PLC gamma 1 with the receptor protein tyrosine kinase. Similarly in lymphocytes, cross-linking of antigen receptors induces the formation of molecular complexes incorporating PLC gamma 1; however, associated kinase activity is thought to be mediated through cytoplasmic protein tyrosine kinase(s). In this report, we generated a fusion protein containing the SH2 domains of human PLC gamma 1 and human IgG1 heavy chain constant region to identify lymphocyte phosphoprotein-binding PLC gamma 1 SH2 domains following cellular activation. As in EGF- or platelet-derived growth factor-stimulated fibroblasts, PLC gamma 1 is coprecipitated in activated lymphocytes, complexed with associated tyrosine-phosphorylated proteins. One of these, a 35/36-kDa protein found prominently in T cells and at lower levels in B cells, bound to the fusion protein in immunoprecipitation experiments. The fusion protein showed lineage restricted association with a 74-kDa phosphoprotein in T cells and a 93-kDa phosphoprotein in B cells. It bound to activated EGF receptor in fibroblasts as expected, and protein tyrosine kinase activity was precipitated from EGF-stimulated cells. However, PLC gamma 1-associated protein tyrosine kinase activity was not detected in activated lymphocytes. These data suggest that lymphocyte PLC gamma 1 SH2-binding proteins are cell lineage specific and may be transiently associated with activated PLC gamma 1.     

A soluble form of the human T cell differentiation antigen CD27 is released after triggering of the TCR/CD3 complex.

The human T cell Ag CD27 belongs to a recently defined family of cell surface receptors, including the nerve growth factor receptor, two distinct tumor necrosis factor receptors, and the B cell specific molecule CD40. On resting T cells, CD27 is a transmembrane homodimer with subunits of 50 to 55 kDa (p55). T cell activation via the TCR/CD3 complex causes a strong enhancement of p55 expression. Concomitantly, an alternative form of the CD27 molecule with a molecular mass of 28 to 32 kDa (p32) appears at the cell surface. With the use of ELISA, we here show that a soluble form of CD27 (sCD27) can be detected in the supernatant of T cells activated with anti-CD3 or combinations of anti-CD2 mAb. Moreover, sCD27 is found in both serum and urine from healthy donors. sCD27, purified from either culture supernatant or urine, has a molecular mass of 28 to 32 kDa and is, according to peptide mapping, structurally homologous to the p55 membrane form of CD27. Quantification of sCD27 levels may be used as a marker for T lymphocyte activation in vivo.     

Normal development but differentially altered proliferative responses of lymphocytes in mice lacking CD81.

CD81 (TAPA-1) is a member of the transmembrane 4 superfamily (TM4SF) which is expressed on the cell surface of most cells of the body throughout their cellular differentiation. It has been recognized in several cell surface complexes of lymphocytes, suggesting that it may have diverse roles in lymphocyte development and activation regulation. Mice with a CD81 null mutation revealed normal T- and conventional B-cell development, although CD19 expression on B cells was dull and B-1 cells were reduced in number. However, both T and B cells of mutant mice exhibited strikingly enhanced proliferation in response to various types of stimuli. Interestingly, while proliferative responses of T cells following T-cell antigen receptor (TCR) engagement was enhanced in the absence of CD81, B-cell proliferation in response to B-cell antigen-receptor (BCR) cross-linking was severely impaired. Despite these altered proliferative responses, both tyrosine phosphorylation and intracellular calcium flux in response to cross-linking of cell surface antigen receptors were normal in mutant mice, reflecting apparently normal initial signaling of antigen receptors. In conclusion, though CD81 is not essential for normal T- and conventional B-cell development, it plays key roles in controlling lymphocyte homeostasis by regulating lymphocyte proliferation in distinct manners, dependent on the context of stimulation.     

Inhibition of T cell antigen receptor-dependent phosphorylation of CD4 in human immunodeficiency virus type 1 infected cells.

Inhibitory effects of human immunodeficiency virus (HIV) on T lymphocyte function have been linked to perturbation of signaling through the T cell antigen receptor-CD3 complex. Comparative biochemical analyses of signaling responses were performed in T cells that were either uninfected or chronically infected with the HIV-1/IIIB strain. Stimulation with antibodies to CD3 triggered both Ca2+ accumulation and phosphoinositide hydrolysis responses that were equivalent in uninfected and infected cells. Treatment with anti-CD3 or with phorbol diester also stimulated serine phosphorylation of CD4 molecules in uninfected T cells. However, phosphorylation of CD4 was not observed after anti-CD3 treatment in HIV-infected T cells despite normal phosphorylation responses to phorbol diester. Identical results were obtained using a T cell line that was infected with an env (gp160/120-) HIV-1 defective variant. These studies indicate that infection with HIV-1 inhibits the activation of protein kinase associated with the T cell receptor-CD3 complex by a mechanism which is independent of viral env protein components.     

DARPP—32的结构,功能及其调节机制

DARPP-32是一种多巴胺（DA）和cAMP调节的磷蛋白，存在于所有接受DA能投射的神经元中，在中枢神经系统的分布与DAD1受体的分布非常一致。DA通过D1受体使DARPP-32第34位苏氨酸磷酸化，磷酸化DARPP-32成为蛋白磷酸酶1（PP-1）的强效抑制剂，在两个不同位点与PP-1相互作用，从而抑制PP-1活性。DARPP-32／PP-1级联反应在调节，如钙通道、电压依赖性钠通道、Na＋,K＋-ATPase和NMDANR1受体的功能等神经元兴奋性过程中起重要作用。DA对DARPP—32的磷酸化状态有双向调节作用，其他许多神经递质亦可调节其磷酸化状态。     

Comparison of the 34,000-Da pp60src substrate and a 38,000-Da phosphoprotein identified by monoclonal antibodies

One of the cellular targets of the pp60src tyrosine kinase is a phosphoprotein with a Mr = 34,000 and an isoelectric point of approximately 7.5 (Radke, K., Gilmore, T., and Martin, G. S. (1980) Cell 21, 821-828; Erikson, E., and Erikson, R. L. (1980) Cell 21, 829-836). We report here the preparation of monoclonal antibodies to partially purified 34-kDa protein and to a heretofore unrecognized phosphoprotein that is not a pp60src target. Two antibodies were initially obtained that recognized phosphoproteins in the Mr = 34,000-39,000 range. One of these antibodies immunoprecipitated a 34,000-Da protein which, on the basis of its molecular mass, phosphorylation state, and isoelectric point, was determined to be the 34-kDa pp60src substrate. The second monoclonal antibody bound to a 38,000-Da nucleolar associated protein, which appeared not to be a target of the pp60src kinase and was found by tryptic analysis to be structurally unrelated to the 34-kDa protein. The monoclonal antibody to the 34-kDa protein coupled to Sepharose CL-4B was used to purify the pp60src substrate to homogeneity in milligram quantities. Both the purified 34-kDa protein and the monoclonal antibody are currently being used in studies aimed at elucidating the structure and function of this pp60src target.     

Nicotine Enhances the Cyclic AMP-Dependent Protein Kinase-Mediated Phosphorylation of α4 Subunits of Neuronal Nicotinic Receptors

Abstract: Studies determined whether α4β2 or α3β2 neuronal nicotinic receptors expressed in Xenopus oocytes are substrates for cyclic AMP-dependent protein kinase (PKA) and whether nicotine affects receptor phosphorylation. The cRNAs for the subunits were coinjected into oocytes, and cells were incubated for 24 h in the absence or presence of nicotine (50 n M for α4β2 and 500 n M for α3β2 receptors). Nicotine did not interfere with the isolation of the receptors. When receptors isolated from oocytes expressing α4β2 receptors were incubated with [γ-³²P]ATP and the catalytic subunit of PKA, separated by electrophoresis, and visualized by autoradiography, a labeled phosphoprotein with the predicted molecular size of the α4 subunit was present. Phosphorylation of α4 subunits of α4β2 receptors increased within the first 5 min of incubation with nicotine and persisted for 24 h. In contrast, receptors isolated from oocytes expressing α3β2 receptors did not exhibit a labeled phosphoprotein corresponding to the size of the α3 subunit. Results suggest that the PKA-mediated phosphorylation of α4 and not α3 subunits may explain the differential inactivation by nicotine of these receptors subtypes expressed in oocytes.     

Profiling of the CD4 receptor complex proteins

Intermolecular complexes produced by the CD4 molecule were studied. To preserve the integrity of weak protein-protein interactions of the CD4 antigen, cells were lysed in a mild nonionic detergent Brij97. Protein constituents of the complex were identified by our previously proposed fluorescence immunoprecipitation assay with subsequent mass spectrometry. In total, 26 proteins associated with CD4 were identified on CEM cells. The CD4 complex included the following major components: tyrosine phosphatase CD45, transferrin receptor CD71, tyrosine kinase Lck, and a lymphocyte phosphatase-associated phosphoprotein LPAP. The CD4 complex also contained some components of cytoskeleton and heat shock proteins. The association between CD4, CD71, and CD45 molecules was confirmed by immunoblotting. The CD4 complexes were not detected on the U937 myeloid cells lacking Lck and LPAP. We attempted to quantitatively characterize the CD4 complex composition.     

The T cell receptor/CD3 complex is composed of at least two autonomous transduction modules.

Recent studies have demonstrated that the CD3-zeta subunit of the T cell antigen receptor (TCR) complex is involved in signal transduction. However, the function of the remaining invariant subunits, CD3-gamma, -delta, and epsilon, is still poorly understood. To examine their role in TCR function, we have constructed TCR/CD3 complexes devoid of functional zeta subunit and showed that they are still able to trigger the production of interleukin-2 in response to antigen or superantigen. These data, together with previous results, indicate that the TCR/CD3 complex is composed of at least two parallel transducing units, made of the gamma delta epsilon and zeta chains, respectively. Furthermore, the analysis of partially truncated zeta chains has led us to individualize a functional domain that may have constituted the building block of most of the transducing subunits associated with antigen receptors and some Fc receptors.