MAP kinase phosphatase as a locus of flexibility in a mitogen-activated protein kinase signaling network

Intracellular signaling networks receive and process information to control cellular machines. The mitogen-activated protein kinase (MAPK) 1,2/protein kinase C (PKC) system is one such network that regulates many cellular machines, including the cell cycle machinery and autocrine/paracrine factor synthesizing machinery. We used a combination of computational analysis and experiments in mouse NIH-3T3 fibroblasts to understand the design principles of this controller network. We find that the growth factor-stimulated signaling network containing MAPK 1, 2/PKC can operate with one (monostable) or two (bistable) stable states. At low concentrations of MAPK phosphatase, the system exhibits bistable behavior, such that brief stimulus results in sustained MAPK activation. The MAPK-induced increase in the amounts of MAPK phosphatase eliminates the prolonged response capability and moves the network to a monostable state, in which it behaves as a proportional response system responding acutely to stimulus. Thus, the MAPK 1, 2/PKC controller network is flexibly designed, and MAPK phosphatase may be critical for this flexible response.     

Regulation of chloride channels by protein kinase C in normal and cystic fibrosis airway epithelia

Apical membrane chloride channels control chloride secretion by airway epithelial cells. Defective regulation of these channels is a prominent characteristic of cystic fibrosis. In normal intact cells, activation of protein kinase C (PKC) by phorbol ester either stimulated or inhibited chloride secretion, depending on the physiological status of the cell. In cell-free membrane patches, PKC also had a dual effect: at a high calcium concentration, PKC inactivated chloride channels; at a low calcium concentration, PKC activated chloride channels. In cystic fibrosis cells, PKC-dependent channel inactivation was normal, but activation was defective. Thus it appears that PKC phosphorylates and regulates two different sites on the channel or on an associated membrane protein, one of which is defective in cystic fibrosis.     

A virus-encoded "superantigen" in a retrovirus-induced immunodeficiency syndrome of mice.

The development of an immunodeficiency syndrome of mice caused by a replication-defective murine leukemia virus (MuLV) is paradoxically associated with a rapid activation and proliferation of CD4+ T cells that are dependent on the presence of B cells. The responses of normal spleen cells to B cell lines that express the defective virus indicated that these lines express a cell surface determinant that shares "superantigenic" properties with some microbial antigens and Mls-like self antigens. This antigen elicited a potent proliferative response that was dependent on the presence of CD4+ T cells and was associated with selective expansion of cells bearing V beta 5. This response was markedly inhibited by a monoclonal antibody specific for the MuLV gag-encoded p30 antigen.     

Protein kinase C and regulation of the local competence of Xenopus ectoderm

The limited competence of embryonic tissue to respond to an inductive signal has an essential, regulatory function in embryonic induction. The molecular basis for the competence of Xenopus ectoderm to differentiate into neural tissue was investigated. Dorsal mesoderm or 12-O-tetradecanoyl phorbol-13-acetate (TPA) caused in vivo activation of protein kinase C (PKC) and neural differentiation mainly in dorsal ectoderm and to a lesser extent in ventral ectoderm. These data correlate with the observations that PKC preparations from dorsal and ventral ectoderm differ, the dorsal PKC preparation being more susceptible to activation by TPA and diolein than is the ventral PKC preparation. Monoclonal antibodies against the bovine PKC alpha plus beta or gamma isozymes immunostained dorsal and ventral ectoderm, respectively, which suggests different localizations of PKC isozymes. These results suggest that PKC participates in the establishment of embryonic competence.     

T cell receptor signaling precedes immunological synapse formation

The area of contact between a T cell and an antigen-presenting cell (APC) is known as the immunological synapse. Although its exact function is unknown, one model suggests that it allows for T cell receptor (TCR) clustering and for sustained signaling in T cells for many hours. Here we demonstrate that TCR-mediated tyrosine kinase signaling in na?ve T cells occurred primarily at the periphery of the synapse and was largely abated before mature immunological synapses had formed. These data suggest that many hours of TCR signaling are not required for T cell activation. These observations challenge current ideas about the role of immunological synapses in T cell activation.     

T cell antigen receptor-mediated activation of phospholipase C requires tyrosine phosphorylation

Triggering of the antigen-specific T cell receptor-CD3 complex (TCR-CD3) stimulates a rapid phospholipase C-mediated hydrolysis of inositol phospholipids, resulting in the production of second messengers and in T cell activation and proliferation. The role of tyrosine phosphorylation in these events was investigated with a tyrosine protein kinase (TPK) inhibitor, genistein. At doses that inhibited TPK activity and tyrosine phosphorylation of the TCR zeta subunit, but not phospholipase C activity, genistein prevented TCR-CD3-mediated phospholipase C activation, interleukin-2 receptor expression, and T cell proliferation. These findings indicate that tyrosine phosphorylation is an early and critical event that most likely precedes, and is a prerequisite for, inositol phospholipid breakdown during receptor-mediated T cell activation.     

Reversal of the TCR stop signal by CTLA-4

The coreceptor cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is pivotal in regulating the threshold of signals during T cell activation, although the underlying mechanism is still not fully understood. Using in vitro migration assays and in vivo two-photon laser scanning microscopy, we showed that CTLA-4 increases T cell motility and overrides the T cell receptor (TCR)-induced stop signal required for stable conjugate formation between T cells and antigen-presenting cells. This event led to reduced contact periods between T cells and antigen-presenting cells that in turn decreased cytokine production and proliferation. These results suggest a fundamentally different model of reverse stop signaling, by which CTLA-4 modulates the threshold for T cell activation and protects against autoimmunity.     

Imaging of memory-specific changes in the distribution of protein kinase C in the hippocampus

Activation of protein kinase C (PKC) can mimic the biophysical effects of associative learning on neurons. Furthermore, classical conditioning of the rabbit nictitating membrane (a form of associative learning) produces translocation of PKC activity from the cytosolic to the membrane compartments of the CA1 region of the hippocampus. Evidence is provided here for a significant change in the amount and distribution of PKC within the CA1 cell field of the rabbit hippocampus that is specific to learning. This change is seen at 1 day after learning as focal increments of [3H]phorbol-12,13-dibutyrate binding to PKC in computer-generated images produced from coronal autoradiographs of rabbit brain. In addition, 3 days after learning, the autoradiographs suggest a redistribution of PKC within CA1 from the cell soma to the dendrites.     

Requirement of AMPA receptor GluR2 phosphorylation for cerebellar long-term depression

Cerebellar long-term depression (LTD) is a model of synaptic memory that requires protein kinase C (PKC) activation and is expressed as a reduction in the number of postsynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors. LTD was absent in cultured cerebellar Purkinje cells from mutant mice lacking the AMPA receptor GluR2 subunit and could be rescued by transient transfection with the wild-type GluR2 subunit. Transfection with a point mutant that eliminated PKC phosphorylation of Ser880 in the carboxy-terminal PDZ ligand of GluR2 failed to restore LTD. In contrast, transfection with a point mutant that mimicked phosphorylation at Ser880 occluded subsequent LTD. Thus, PKC phosphorylation of GluR2 Ser880 is a critical event in the induction of cerebellar LTD.     

Identification of a putative regulator of early T cell activation genes

Molecules involved in the antigen receptor-dependent regulation of early T cell activation genes were investigated with the use of functional sequences of the T cell activation-specific enhancer of interleukin-2 (IL-2). One of these sequences forms a protein complex, NFAT-1, specifically with nuclear extracts of activated T cells. This complex appeared 10 to 25 minutes before the activation of the IL-2 gene. Studies with inhibitors of protein synthesis indicated that the time of synthesis of the activator of the IL-2 gene in Jurkat T cells corresponds to the time of appearance of NFAT-1. NFAT-1, or a very similar protein, bound functional sequences of the long terminal repeat (LTR) of the human immunodeficiency virus type 1; the LTR of this virus is known to be stimulated during early T cell activation. The binding site for this complex activated a linked promoter after transfection into antigen receptor-activated T cells but not other cell types. These characteristics suggest that NFAT-1 transmits signals initiated at the T cell antigen receptor.     

Protein kinase C overactivity impairs prefrontal cortical regulation of working memory

The prefrontal cortex is a higher brain region that regulates thought, behavior, and emotion using representational knowledge, operations often referred to as working memory. We tested the influence of protein kinase C (PKC) intracellular signaling on prefrontal cortical cognitive function and showed that high levels of PKC activity in prefrontal cortex, as seen for example during stress exposure, markedly impair behavioral and electrophysiological measures of working memory. These data suggest that excessive PKC activation can disrupt prefrontal cortical regulation of behavior and thought, possibly contributing to signs of prefrontal cortical dysfunction such as distractibility, impaired judgment, impulsivity, and thought disorder.     

Cell cycle-dependent coupling of the calcitonin receptor to different G proteins.

Calcitonin is a calcium regulating peptide hormone with binding sites in kidney and bone as well as in the central nervous system. The mechanisms of signal transduction by calcitonin receptors were studied in a pig kidney cell line where the hormone was found to regulate sodium pumps. Calcitonin receptors activated the cyclic adenosine monophosphate (cAMP) or the protein kinase C (PKC) pathways. The two transduction pathways required guanosine triphosphate (GTP)-binding proteins (G proteins) (the choleratoxin sensitive Gs and the pertussis toxin sensitive Gi, respectively) and led to opposite biological responses. Moreover, selective activation of one or the other pathway was cell cycle-dependent. Therefore, calcitonin may induce different biological responses in target cells depending on their positions in the cell cycle. Such a modulation of ligand-induced responses could be of importance in rapidly growing cell populations such as during embryogenesis, growth, and tumor formation.     

A LAT mutation that inhibits T cell development yet induces lymphoproliferation

Mice homozygous for a single tyrosine mutation in LAT (linker for activation of T cells) exhibited an early block in T cell maturation but later developed a polyclonal lymphoproliferative disorder and signs of autoimmune disease. T cell antigen receptor (TCR)-induced activation of phospholipase C-gamma1 (PLC-gamma1) and of nuclear factor of activated T cells, calcium influx, interleukin-2 production, and cell death were reduced or abrogated in T cells from LAT mutant mice. In contrast, TCR-induced Erk activation was intact. These results identify a critical role for integrated PLC-gamma1 and Ras-Erk signaling through LAT in T cell development and homeostasis.     

Identification of a T helper cell-derived lymphokine that activates resting T lymphocytes

A novel lymphokine with apparent molecular size of 10 to 12 kilodaltons is secreted from helper T cell clones within hours after cross-linking their T cell antigen-MHC (major histocompatibility complex) receptors (T3-Ti). This lymphokine, termed interleukin-4A (IL-4A), stimulates resting lymphocytes by binding to a surface component (or components) of the alternative T11 pathway and subsequently by inducing interleukin-2 (IL-2) receptors. The activation process is neither dependent on antigen specificities of the recruited population or the presence of macrophages. It appears, therefore, that IL-4A is a mediator involved in amplifying the T cell immune response.     

Nuclear magnetic resonance studies and water "ordering" in the crystalline lens

Nuclear magnetic resonance studies of the relaxation times of the water in the crystalline lens show that, as in all interfacial systems, these parameters are markedly reduced from their values in pure water, that T(2) is less than T(1), and that both depend on water content. Determination of diffusion coefficients and studies on physiologically inert lenses indicate that reduced relaxation times do not provide direct evidence for ordering of the bulk of the cell water.     

Vital involvement of a natural killer cell activation receptor in resistance to viral infection

Natural killer (NK) cells are lymphocytes that can be distinguished from T and B cells through their involvement in innate immunity and their lack of rearranged antigen receptors. Although NK cells and their receptors were initially characterized in terms of tumor killing in vitro, we have determined that the NK cell activation receptor, Ly-49H, is critically involved in resistance to murine cytomegalovirus in vivo. Ly-49H requires an immunoreceptor tyrosine-based activation motif (ITAM)-containing transmembrane molecule for expression and signal transduction. Thus, NK cells use receptors functionally resembling ITAM-coupled T and B cell antigen receptors to provide vital innate host defense.     

Synergism between HIV gp120 and gp120-specific antibody in blocking human T cell activation

The human immunodeficiency virus (HIV) binds to CD4-positive cells through interaction of its envelope glycoprotein (gp120) with the CD4 molecule. CD4 is a prominent immunoregulatory molecule, and chronic exposure to antibody against CD4 (anti-CD4) has been shown to cause immunodeficiency in mice. T cell-dependent in vitro immune responses can also be inhibited by anti-CD4. Experimental findings reported here indicate that CD4-bound gp120 attracts gp120-specific antibodies derived from the blood of HIV-seropositive individuals to form a trimolecular complex with itself and CD4. Thus targeted to CD4, the gp120-specific antibody functions as an antibody to CD4; it cross-links and modulates the CD4 molecules and suppresses the activation of T cells as measured by mobilization of intracellular calcium (Ca2i+). The synergism between gp120 and anti-gp120 in blocking T cell activation occurs at low concentrations of both components. Neither gp120 nor anti-gp120 inhibits T cell activation by itself in the concentrations tested.     

Induction of T helper type 2 immunity by a point mutation in the LAT adaptor

The transmembrane protein LAT (linker for activation of T cells) couples the T cell receptor (TCR) to downstream signaling effectors. Mice homozygous for a mutation of a single LAT tyrosine residue showed impeded T cell development. However, later they accumulated polyclonal helper T (TH) cells that chronically produced type 2 cytokines in large amounts. This exaggerated TH2 differentiation caused tissue eosinophilia and massive maturation of plasma cells secreting to immunoglobulins of the E and G1 isotypes. This paradoxical phenotype establishes an unanticipated inhibitory function for LAT that is critical for the differentiation and homeostasis of TH cells.