The AMPA receptor interacts with and signals through the protein tyrosine kinase Lyn

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. The ionotropic glutamate receptors are classified into two groups, NMDA (N-methyl-D-aspartate) receptors and AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate) receptors. The AMPA receptor is a ligand-gated cation channel that mediates the fast component of excitatory postsynaptic currents in the central nervous system. Here we report that AMPA receptors function not only as ion channels but also as cell-surface signal transducers by means of their interaction with the Src-family non-receptor protein tyrosine kinase Lyn. In the cerebellum, Lyn is physically associated with the AMPA receptor and is rapidly activated following stimulation of the receptor. Activation of Lyn is independent of Ca2+ and Na+ influx through AMPA receptors. As a result of activation of Lyn, the mitogen-activated protein kinase (MAPK) signalling pathway is activated, and the expression of brain-derived neurotrophic factor (BDNF) messenger RNA is increased in a Lyn-kinase-dependent manner. Thus, AMPA receptors generate intracellular signals from the cell surface to the nucleus through the Lyn-MAPK pathway, which may contribute to synaptic plasticity by regulating the expression of BDNF.     

Differential regulation of focal adhesion kinase and mitogen-activated protein kinase tyrosine phosphorylation during insulin-like growth factor-I-mediated cytoskeletal reorganization

In SH-SY5Y human neuroblastoma cells, insulin-like growth factor (IGF)-I mediates membrane ruffling and growth cone extension. We have previously shown that IGF-I activates the tyrosine phosphorylation of focal adhesion kinase (FAK) and extracellular signal-regulated protein kinase (ERK) 2. In the current study, we examined which signaling pathway underlies IGF-I-mediated FAK phosphorylation and cytoskeletal changes and determined if an intact cytoskeleton was required for IGF-I signaling. Treatment of SH-SY5Y cells with cytochalasin D disrupted the actin cytoskeleton and prevented any morphological changes induced by IGF-I. Inhibitors of phosphatidylinositol 3-kinase (PI 3-K) blocked IGF-I-mediated changes in the actin cytoskeleton as measured by membrane ruffling. In contrast, PD98059, a selective inhibitor of ERK kinase, had no effect on IGF-I-induced membrane ruffling. In parallel with effects on the actin cytoskeleton, cytochalasin D and PI 3-K inhibitors blocked IGF-I-induced FAK tyrosine phosphorylation, whereas PD98059 had no effect. It is interesting that cytochalasin D did not block IGF-I-induced ERK2 tyrosine phosphorylation. Therefore, it is likely that FAK and ERK2 tyrosine phosphorylations are regulated by separate pathways during IGF-I signaling. Our study suggests that integrity as well as dynamic motility of the actin cytoskeleton mediated by PI 3-K is required for IGF-I-induced FAK tyrosine phosphorylation, but not for ERK2 activation.     

NCAM140 interacts with the focal adhesion kinase p125(fak) and the SRC-related tyrosine kinase p59(fyn)

Axonal growth cones respond to adhesion molecules and extracellular matrix components by rapid morphological changes and growth rate modification. Neurite outgrowth mediated by the neural cell adhesion molecule (NCAM) requires the src family tyrosine kinase p59(fyn) in nerve growth cones, but the molecular basis for this interaction has not been defined. The NCAM140 isoform, which is found in migrating growth cones, selectively co-immunoprecipitated with p59(fyn) from nonionic detergent (Brij 96) extracts of early postnatal mouse cerebellum and transfected rat B35 neuroblastoma and COS-7 cells. p59(fyn) did not associate significantly with the NCAM180 isoform, which is found at sites of stable neural cell contacts, or with the glycophosphatidylinositol-linked NCAM120 isoform. pp60(c-)src, a tyrosine kinase that promotes neurite growth on the neuronal cell adhesion molecule L1, did not interact with any NCAM isoform. Whereas p59(fyn) was constitutively associated with NCAM140, the focal adhesion kinase p125(fak), a nonreceptor tyrosine kinase known to mediate integrin-dependent signaling, became recruited to the NCAM140-p59(fyn) complex when cells were reacted with antibodies against the extracellular region of NCAM. Treatment of cells with a soluble NCAM fusion protein or with NCAM antibodies caused a rapid and transient increase in tyrosine phosphorylation of p125(fak) and p59(fyn). These results suggest that NCAM140 binding interactions at the cell surface induce the assembly of a molecular complex of NCAM140, p125(fak), and p59(fyn) and activate the catalytic function of these tyrosine kinases, initiating a signaling cascade that may modulate growth cone migration.     

The adaptor protein Nck links receptor tyrosine kinases with the serine-threonine kinase Pak1

Nck is an adaptor protein composed of a single SH2 domain and three SH3 domains. Upon growth factor stimulation, Nck is recruited to receptor tyrosine kinases via its SH2 domain, probably initiating one or more signaling cascades. In this report, we show that Nck is bound in living cells to the serine-threonine kinase Pak1. The association between Nck and Pak1 is mediated by the second SH3 domain of Nck and a proline-rich sequence in the amino terminus of Pak1. We also show that Pak1 is recruited by activated epidermal growth factor (EGF) and platelet-derived growth factor receptors. Moreover, Pak1 kinase activity is increased in response to EGF in HeLa cells transfected with human Pak1, and the kinase activity was enhanced when Nck was co-transfected. It is concluded that Nck links receptor tyrosine kinases with Pak1 and is probably involved in targeting and regulation of Pak1 activity.     

Constitutive tyrosine phosphorylation of the T-cell receptor (TCR) zeta subunit: regulation of TCR-associated protein tyrosine kinase activity by TCR zeta

The T-cell receptor (TCR) zeta subunit is an important component of the TCR complex, involved in signal transduction events following TCR engagement. In this study, we showed that the TCR zeta chain is constitutively tyrosine phosphorylated to similar extents in thymocytes and lymph node T cells. Approximately 35% of the tyrosine-phosphorylated TCR zeta (phospho zeta) precipitated from total cell lysates appeared to be surface associated. Furthermore, constitutive phosphorylation of TCR zeta in T cells occurred independently of antigen stimulation and did not require CD4 or CD8 coreceptor expression. In lymph node T cells that constitutively express tyrosine-phosphorylated TCR zeta, there was a direct correlation between surface TCR-associated protein tyrosine kinase (PTK) activity and expression of phospho zeta. TCR stimulation of these cells resulted in an increase in PTK activity that coprecipitated with the surface TCR complex and a corresponding increase in the levels of phospho zeta. TCR ligations also contributed to the detection of several additional phosphoproteins that coprecipitated with surface TCR complexes, including a 72-kDa tyrosine-phosphorylated protein. The presence of TCR-associated PTK activity also correlated with the binding of a 72-kDa protein, which became tyrosine phosphorylated in vitro kinase assays, to tyrosine phosphorylated TCR zeta. The cytoplasmic region of the TCR zeta chain was synthesized, tyrosine phosphorylated, and conjugated to Sepharose beads. Only tyrosine-phosphorylated, not nonphosphorylated, TCR zeta beads were capable of immunoprecipitating the 72-kDa protein from total cell lysates. This 72-kDa protein is likely the murine equivalent of human PTK ZAP-70, which has been shown to associate specifically with phospho zeta. These results suggest that TCR-associated PTK activity is regulated, at least in part, by the tyrosine phosphorylation status of TCR zeta.     

The XRCC4 gene product is a target for and interacts with the DNA-dependent protein kinase

The gene product of XRCC4 has been implicated in both V(D)J recombination and the more general process of double strand break repair (DSBR). To date its role in these processes is unknown. Here, we describe biochemical characteristics of the murine XRCC4 protein. XRCC4 expressed in insect cells exists primarily as a disulfide-linked homodimer, although it can also form large multimers. Recombinant XRCC4 is phosphorylated during expression in insect cells. XRCC4 phosphorylation in Sf9 cells occurs on serine, threonine, and tyrosine residues. We also investigated whether XRCC4 interacts with the other factor known to be requisite for both V(D)J recombination and DSBR, the DNA-dependent protein kinase. We report that XRCC4 is an efficient in vitro substrate of DNA-PK and another unidentified serine/ threonine protein kinase(s). Both DNA-PK dependent and independent phosphorylation of XRCC4 in vitro occurs only on serine and threonine residues within the COOH-terminal 130 amino acids, a region of the molecule that is not absolutely required for XRCC4's DSBR function. Finally, recombinant XRCC4 facilitates Ku binding to DNA, promoting assembly of DNA-PK and complexing with DNA-PK bound to DNA. These data are consistent with the hypothesis that XRCC4 functions as an alignment factor in the DNA-PK complex.     

msh may play a conserved role in dorsoventral patterning of the neuroectoderm and mesoderm

Many of the mechanisms that govern the patterning of the Drosophila neuroectoderm and mesoderm are still unknown. Here we report the sequence, expression, and regulation of the homeobox gene msh, which is likely to play an important role in the early patterning events of these two tissue primordia. msh expression is first observed in late blastoderm embryos and occurs in longitudinal bands of cells that are fated to become lateral neuroectoderm. This expression is under the control of dorsoventral axis-determination genes and depends on dpp-mediated repression in the dorsal half of the embryo and on fib-(EGF-) mediated repression ventrally. The bands of msh expression define the cells that will form the lateral columns of proneural gene expression and give rise to the lateral row of SI neuroblasts. This suggests that msh may be one of the upstream regulators of the achaete-scute (AS-C) genes and may play a role that is analogous to that of the homeobox gene vnd/NK2 in the medial sector of the neuroectoderm. During neuroblast segregation, msh expression is maintained in a subset of neuroblasts, indicating that msh, like vnd/NK2, could function in both dorsoventral patterning of the neuroectoderm and neuroblast specification. The later phase of msh expression that occurs after the first wave of neuroblast segregation in defined ectodermal and mesodermal clusters of cells points to similar roles of msh in patterning and cell fate specification of the peripheral nervous system, dorsal musculature, and the fat body. A comparison of the expression patterns of the vertebrate homologs of msh, vnd/NK2, and AS-C genes reveals striking similarities in dorsoventral patterning of the Drosophila and vertebrate neuroectoderm and indicates that genetic circuitries in neural patterning are evolutionarily conserved.     

Huntingtin-associated protein 1 (HAP1) interacts with the p150Glued subunit of dynactin

Huntington's disease (HD) is an inherited neurodegenerative disease caused by expansion of a polyglutamine repeat in the HD protein huntingtin. Huntingtin's localization within the cell includes an association with cytoskeletal elements and vesicles. We previously identified a protein (HAP1) which binds to huntingtin in a glutamine repeat length-dependent manner. We now report that HAP1 interacts with cytoskeletal proteins, namely the p150 Glued subunit of dynactin and the pericentriolar protein PCM-1. Structural predictions indicate that both HAP1 and the interacting proteins have a high probability of forming coiled coils. We examined the interaction of HAP1 with p150 Glued . Binding of HAP1 to p150 Glued (amino acids 879-1150) was confirmed in vitro by binding of p150 Glued to a HAP1-GST fusion protein immobilized on glutathione-Sepharose beads. Also, HAP1 co-immunoprecipitated with p150 Glued from brain extracts, indicating that the interaction occurs in vivo . Like HAP1, p150 Glued is highly expressed in neurons in brain and both proteins are enriched in a nerve terminal vesicle-rich fraction. Double label immunofluorescence experiments in NGF-treated PC12 cells using confocal microscopy revealed that HAP1 and p150 Glued partially co-localize. These results suggest that HAP1 might function as an adaptor protein using coiled coils to mediate interactions among cytoskeletal, vesicular and motor proteins. Thus, HAP1 and huntingtin may play a role in vesicle trafficking within the cell and disruption of this function could contribute to the neuronal dysfunction and death seen in HD.     

The wheat poly(A)-binding protein functionally complements pab1 in yeast

Poly(A)-binding protein (PAB) binds to the poly(A) tail of most eukaryotic mRNAs and influences its translational efficiency as well as its stability. Although the primary structure of PAB is well conserved in eukaryotes, its functional conservation across species has not been extensively investigated. In order to determine whether PAB from a monocot plant species could function in yeast, a protein characterized as having PAB activity was purified from wheat and a cDNA encoding for PAB was isolated from a wheat seedling expression library. Wheat PAB (72 kDa as estimated by SDS/PAGE and a theoretical mass of 70 823 Da as determined from the cDNA) was present in multiple isoforms and exhibited binding characteristics similar to that determined for yeast PAB. Comparison of the wheat PAB protein sequence with PABs from yeast and other species revealed that wheat PAB contained the characteristic features of all PABs, including four RNA binding domains each of which contained the conserved RNP1 and RNP2 sequence motifs. The wheat PAB cDNA functionally complemented a pab1 mutant in yeast suggesting that, although the amino acid sequence of wheat PAB is only 47% conserved from that of yeast PAB, this monocot protein can function in yeast.     

The type II IL-1 receptor interacts with the IL-1 receptor accessory protein: a novel mechanism of regulation of IL-1 responsiveness

IL-1 binds to two types of receptors on the cell membrane, of which only type I (IL-1RI) transduces signals in concert with the coreceptor IL-1 receptor accessory protein (IL-1RAcP) while type II (IL-1RII) allegedly functions solely as ligand sink and decoy receptor without participating in IL-1 signaling. To investigate the regulatory role of IL-1RII on IL-1 responsiveness, a chimeric receptor encompassing the extracellular and transmembrane portions of IL-1RII and the cytoplasmic signal-transducing domain of IL-1RI was transfected into two murine EL-4-derived sublines that do or do not express IL-1RAcP, respectively. The chimeric receptor was able to transduce the IL-1 signal and induce IL-2 production only in the cell line which expressed IL-1RAcP, suggesting effective interaction between the extracellular domains of IL-1RII and IL-1RAcP in the presence of IL-1. The physical association of ligated IL-1RII with IL-1RAcP was proven by crosslinking experiments with radio-iodinated IL-1 and subsequent immunoprecipitations in normal human B cells and in EL-4 D6/76 cells transiently cotransfected with IL-1RII and IL-1RAcP, respectively. Based on these findings, it is proposed that upon IL-1 binding IL-1RII can recruit IL-1RAcP into a nonfunctional trimeric complex and thus modulate IL-1 signaling by subtracting the coreceptor molecule from the signaling IL-1RI. In this novel mechanism of coreceptor competition, the ratio between IL-1RII and IL-1RI becomes the central factor in determining the IL-1 responsiveness of a cell and the availability of IL-1RAcP becomes limiting for effective IL-1 signaling.     

Altered regulation of L-type channels by protein kinase C and protein tyrosine kinases as a pathophysiologic effect in retinal degeneration

The effect of protein tyrosine kinases (PTK) on L-type calcium channels in cultured retinal pigmented epithelium (RPE) from rats with retinal dystrophy was investigated. Barium currents through Bay K 8644 (10(-6) M) sensitive L-type channels were measured using the patch-clamp technique. The current density of L-type currents is twice as high and the inactivation time constants are much slower than in cells from nondystrophic control rats. Application of the PTK blockers genistein, lavendustin A, and herbimycin A (all 5 x 10(-6) M) led to an increase of L-type currents. Intracellular application of pp60c-src (30 U/ml) via the patch pipette led to a transient decrease of L-type currents. The protein kinase A (PKA) and PKG blocker H9 (10(-6) M) showed no effect on L-type currents. However, the protein kinase C blocker chelerythrine (10(-5) M) reduced these currents. Up-regulation of PKC by 10(-6) M 4beta-phorbol-12 myristate-13 acetate (PMA) led to a decrease of L-type currents. Additional application of genistein led to a further decrease of these currents. However, intracellular application of pp60(c-src) in PMA-treated cells led to a transient increase of L-type currents. Investigating the calcium response to bFGF application showed that RPE cells from RCS rats used different pathways than control RPE cells to increase cytosolic free calcium. This different pathway does not involve the activation of L-type channels. The present study with RPE cells from rats with retinal dystrophy shows a changed integration of PTK and PKC in channel regulation. Considering the altered response to bFGF in RCS-RPE cells, this disturbed regulation of L-type channels by tyrosine kinases is involved in the etiology of retinal degeneration in RCS rats.     

A role for Bruton's tyrosine kinase (Btk) in platelet activation by collagen

Bruton's tyrosine kinase (Btk) is essential for normal B-cell receptor signalling. The lack of expression of functional Btk in humans leads to the B-cell deficiency X-linked agammaglobulinaemia (XLA). We report here that Btk is also important for signalling via the collagen receptor glycoprotein VI (GPVI) in platelets. GPVI is coupled to the Fc receptor gamma chain (FcRgamma). The FcRgamma-chain contains a consensus sequence known as the immune-receptor tyrosine-based activation motif (ITAM). Tyrosine phosphorylation of the ITAM upon GPVI stimulation is the initial step in the regulation of phospholipase C gamma2 (PLCgamma2) isoforms via the tyrosine kinase p72(Syk) (Syk) in platelets. Here we show that collagen and a collagen-related peptide (CRP), which binds to GPVI but does not bind to the integrin alpha2beta1, induced Btk tyrosine phosphorylation in platelets. Aggregation, dense granule secretion and calcium mobilisation were significantly diminished but not completely abolished in platelets from XLA patients in response to collagen and CRP. These effects were associated with a reduction in tyrosine phosphorylation of PLCgamma2. In contrast, aggregation and secretion stimulated by thrombin in Btk-deficient platelets were not significantly altered. Our results demonstrate that Btk is important for collagen signalling via GPVI, but is not essential for thrombin-mediated platelet activation.     

ARG1 (altered response to gravity) encodes a DnaJ-like protein that potentially interacts with the cytoskeleton

Gravitropism allows plant organs to direct their growth at a specific angle from the gravity vector, promoting upward growth for shoots and downward growth for roots. Little is known about the mechanisms underlying gravitropic signal transduction. We found that mutations in the ARG1 locus of Arabidopsis thaliana alter root and hypocotyl gravitropism without affecting phototropism, root growth responses to phytohormones or inhibitors of auxin transport, or starch accumulation. The positional cloning of ARG1 revealed a DnaJ-like protein containing a coiled-coil region homologous to coiled coils found in cytoskeleton-interacting proteins. These data suggest that ARG1 participates in a gravity-signaling process involving the cytoskeleton. A combination of Northern blot studies and analysis of ARG1-GUS fusion-reporter expression in transgenic plants demonstrated that ARG1 is expressed in all organs. Ubiquitous ARG1 expression in Arabidopsis and the identification of an ortholog in Caenorhabditis elegans suggest that ARG1 is involved in other essential processes.     

The Cdc14 phosphatase is functionally associated with the Dbf2 protein kinase in Saccharomyces cerevisiae

The Saccharomyces cerevisiae Cdc14 protein phosphatase and Dbf2 protein kinase have been implicated to act during late M phase, but their functions are not known. We report here that CDC14 is a low-copy suppressor of the dbf2-2 mutation at 37 degrees C. The kinase activity of Dbf2 accumulated at a high level, in vivo, during a cdc14 arrest and was also much higher in cdc14 mutant cells at the permissive temperature of growth, therefore in cycling mutant cells than in cycling wild-type cells. This correlated with the accumulation of the more slowly migrating form of Dbf2, previously shown to correspond to the hyperphosphorylated form of the protein. The finding that the dbf2-2 mutation could be rescued following overproduction of catalytically inactive forms of Cdc14 suggested that the control of Dbf2 activity by Cdc14 might be only indirect and independent of Cdc14 phosphatase activity. However, it was found that Cdc14 could form oligomers within the cell, thus leaving open the possibility that catalytically inactive Cdc14 might associate with wild-type Cdc14 and rescue dbf2-2 in a phosphatase-dependent manner. We confirmed that overexpression of CDC14 could rescue mutations in CDC15, which encodes another kinase also implicated to act in late M phase. Cells of a cdc15-2 dbf2-2 double mutant died at temperatures much lower than did either single mutant, whereas there was only a slight additive phenotype in the cdc14-1 dbf2-2 and cdc14-1 cdc15-2 double mutant cells. Finally, functional association between Cdc14 and Dbf2 (and also Cdc15) was confirmed by the finding that the cdc14, dbf2 and cdc15 mutations could be partially rescued by the addition of 1.2 M sorbitol to the culture medium. Our data are the first to demonstrate a functional link between Cdc14 and Dbf2 based on both biochemical and genetic information.