Identification of in vivo phosphorylation sites required for protein kinase D activation

Protein kinase D (PKD) is activated by phosphorylation in intact cells stimulated by phorbol esters, cell permeant diacylglycerols, bryostatin, neuropeptides, and growth factors, but the critical activating residues in PKD have not been identified. Here, we show that substitution of Ser744 and Ser748 with alanine (PKD-S744A/S748A) completely blocked PKD activation induced by phorbol-12,13-dibutyrate (PDB) treatment of intact cells as assessed by autophosphorylation and exogenous syntide-2 peptide substrate phosphorylation assays. Conversely, replacement of both serine residues with glutamic acid (PKD-S744E/S748E) markedly increased basal activity (7.5-fold increase compared with wild type PKD). PKD-S744E/S748E mutant was only slightly further stimulated by PDB treatment in vivo, suggesting that phosphorylation of these two sites induces maximal PKD activation. Two-dimensional tryptic phosphopeptide analysis obtained from PKD mutants immunoprecipitated from 32P-labeled transfected COS-7 cells showed that two major spots present in the PDB-stimulated wild type PKD or the kinase-dead PKD-D733A phosphopeptide maps completely disappeared in the kinase-deficient triple mutant PKD-D733A/S744E/S748E. Our results indicate that PKD is activated by phosphorylation of residues Ser744 and Ser748 and thus provide the first example of a non-RD kinase that is up-regulated by phosphorylation of serine/threonine residues within the activation loop.     

LAT is required for TCR-mediated activation of PLCgamma1 and the Ras pathway

In this study, we present the further characterization of a mutant Jurkat T cell line, J.CaM2, that is defective in TCR-mediated signal transduction. Although initial TCR-mediated signaling events such as the inducible tyrosine phosphorylation of the TCR-zeta chain and ZAP-70 are intact in J.CaM2, subsequent events, including increases in intracellular calcium, Ras activation, and IL-2 gene expression are defective. Subsequent analysis of J.CaM2 demonstrated a severe deficiency in pp36/LAT expression, a recently cloned adaptor protein implicated in TCR signaling. Importantly, reexpression of LAT in J.CaM2 restored all aspects of TCR signaling. These results demonstrate a necessary and exclusive role for LAT in T cell activation.     

Tyrosine phosphorylation is required for Fc receptor-mediated phagocytosis in mouse macrophages

Although Fc receptor-mediated phagocytosis is accompanied by a variety of transmembrane signaling events, not all signaling events are required for particle ingestion. For example, Fc receptor-mediated phagocytosis in mouse inflammatory macrophages (Di Virgilio, F., B. C. Meyer, S. Greenberg, and S. C. Silverstein. 1988. J. Cell Biol. 106:657; Greenberg, S., J. El Khoury, F. Di Virgilio, and S. C. Silverstein. 1991. J. Cell Biol. 113:757) and neutrophils (Della Bianca, V., M. Grzeskowiak, and F. Rossi. 1990. J. Immunol. 144:1411) occurs in the absence of cytosolic calcium transients. We sought to identify transmembrane signaling events that are essential for phagocytosis. Here we show that tyrosine phosphorylation is an early event after Fc receptor ligation in mouse inflammatory macrophages, and that the formation of tyrosine phosphoproteins coincides temporally with the appearance of F-actin beneath phagocytic cups. The distribution of tyrosine phosphoproteins that accumulated beneath phagocytic cups was punctate and corresponded to areas of high ligand density on the surface of the antibody-coated red blood cells, which provided the phagocytic stimulus. A tyrosine kinase inhibitor, genistein, but not several inhibitors of protein kinase C, blocked the appearance of tyrosine phosphoproteins as assessed by immunofluorescence, the focal accumulation of F-actin beneath immunoglobulin G-opsonized particles, and the ingestion of these particles as well. We suggest that tyrosine phosphorylation is a critical signaling event that underlies Fc receptor-mediated phagocytosis in mouse macrophages, and is necessary for the engulfment per se.     

The exonuclease activity of HSV-1 UL12 is required for in vivo function

The herpes simplex virus type 1 (HSV-1) UL12 gene encodes an alkaline pH-dependent deoxyribonuclease termed alkaline nuclease. A recombinant UL12 knockout mutant, AN-1, is severely compromised for growth, and analysis of this mutant suggests that UL12 plays a role in processing complex DNA replication intermediates (R. Martinez, R. T. Sarisky, P. C. Weber, and S. K. Weller, (1996) J. Virol. 70, 2075-2085). This processing step may be required for the generation of capsids that are competent for egress from the nucleus to the cytoplasm. In this report, we address the question of whether the AN-1 growth phenotype is due to the loss of UL12 catalytic activity. We constructed two point mutations in a highly conserved region (motif II) of UL12 and purified wild-type and mutant enzymes from a baculovirus expression system. Both mutant proteins are stable, soluble, and competent for correct nuclear localization, suggesting that they have retained an intact global conformation. Neither mutant protein, however, exhibits exonuclease activity. In order to examine the in vivo effects of these mutations, we determined whether expression of mutant proteins from amplicon plasmids could complement AN-1. While the wild-type plasmid complements the growth of the null mutant, neither UL12 mutant can do so. Loss of exonuclease activity therefore correlates with loss of in vivo function.     

Glycine-receptor activation is required for receptor clustering in spinal neurons

The ability of nerve cells to receive up to several thousands of synaptic inputs from other neurons provides the anatomical basis for information processing in the vertebrate brain. The formation of functional synapses involves selective clustering of neurotransmitter receptors at presumptive postsynaptic regions of the neuronal plasma membrane. Receptor-associated proteins are believed to be crucial for this process. In spinal neurons, synaptic targeting of the inhibitory glycine receptor (GlyR) depends on the expression of the anchoring protein gephyrin. Here we show that the competitive GlyR antagonist strychnine and L-type Ca2+-channel blockers inhibit the accumulation of GlyR and gephyrin at postsynaptic membrane areas in cultured rat spinal neurons. Our data are consistent with a model in which GlyR activation that results in Ca2+ influx is required for the clustering of gephyrin and GlyR at developing postsynaptic sites. Similar activity-driven mechanisms may be of general importance in synaptogenesis.     

Bacterial invasion is not required for activation of NF-kappaB in enterocytes

Pathogenic enteric microorganisms induce the NF-kappaB-dependent expression of proinflammatory genes in intestinal epithelial cells. The purpose of the present study was to clarify the contribution of microbial invasion to the degradation of the regulatory protein Ikappa Balpha and the subsequent activation of NF-kappaB in cultured intestinal epithelial cells. Caco-2BBe cells were incubated with Salmonella dublin, Salmonella typhimurium, or a weakly invasive strain of E. coli. S. dublin and S. typhimurium (10(7) organisms/ml) induced equivalent concentration-dependent gel mobility shifts of an NF-kappaB consensus sequence that was preceded by Ikappa Balpha degradation. E. coli (10(7) organisms/ml) did not induce Ikappa Balpha degradation or NF-kappaB translocation. Pretreatment with cytochalasin D blocked invasion of all three strains but had no effect on Ikappa Balpha degradation or NF-kappaB activation. S. dublin and S. typhimurium adhered to Caco-2BBe cells 3- to 10-fold more than E. coli. NF-kappaB activation was prevented by physical separation of S. dublin from Caco-2BBe cells by a 0. 4-micrometers-pore-size filter. Our results imply that bacterial adhesion, rather than invasion or release of a secreted factor, is sufficient to induce IkappaBalpha degradation and NF-kappaB activation in intestinal epithelial cells. Our data suggest that strategies to reduce enteric inflammation should be directed to the reduction of bacterial enterocyte adhesion.     

Phosphatidylinositol 3-kinase is required for integrin-stimulated AKT and Raf-1/mitogen-activated protein kinase pathway activation

Cell attachment to fibronectin stimulates the integrin-dependent interaction of p85-associated phosphatidylinositol (PI) 3-kinase with integrin-dependent focal adhesion kinase (FAK) as well as activation of the Ras/mitogen-activated protein (MAP) kinase pathway. However, it is not known if this PI 3-kinase-FAK interaction increases the synthesis of the 3-phosphorylated phosphoinositides (3-PPIs) or what role, if any, is played by activated PI 3-kinase in integrin signaling. We demonstrate here the integrin-dependent accumulation of the PI 3-kinase products, PI 3,4-bisphosphate [PI(3,4)P2] and PI(3,4,5)P3, as well as activation of AKT kinase, a serine/threonine kinase that can be stimulated by binding of PI(3,4)P2. The PI 3-kinase inhibitors wortmannin and LY294002 significantly decreased the integrin-induced accumulation of the 3-PPIs and activation of AKT kinase, without having significant effects on the levels of PI(4,5)P2 or tyrosine phosphorylation of paxillin. These inhibitors also reduced cell adhesion/spreading onto fibronectin but had no effect on attachment to polylysine. Interestingly, integrin-mediated Erk-2, Mek-1, and Raf-1 activation, but not Ras-GTP loading, was inhibited at least 80% by wortmannin and LY294002. In support of the pharmacologic results, fibronectin activation of Erk-2 and AKT kinases was completely inhibited by overexpression of a dominant interfering p85 subunit of PI 3-kinase. We conclude that integrin-mediated adhesion to fibronectin results in the accumulation of the PI 3-kinase products PI(3,4)P2 and PI(3,4,5)P3 as well as the PI 3-kinase-dependent activation of the kinases Raf-1, Mek-1, Erk-2, and AKT and that PI 3-kinase may function upstream of Raf-1 but downstream of Ras in integrin activation of Erk-2 MAP and AKT kinases.     

BDNF is required for the normal development of taste neurons in vivo

The vallate gustatory epithelium of neonatal trkB null mutant mice (-/-) lacked innervation. This prompted the evaluation of null mutant mice corresponding to the three neurotrophin ligands for tyrosine kinase receptor B (TrkB): brain-derived neurotrophic factor (BDNF), neurotrophin (NT)3, NT4. The vallate gustatory epithelium of nt3-/- mice and of nt4-/- mice appeared normal. Only bdnf-/- mice had a vallate papilla that was stunted, sparsely innervated, and lacked up to 98% of its taste buds. All three defects persisted. For example, the vallate papilla of 12-day-old bdnf-/- mice remained markedly less well innervated than the vallate of 7-day-old or newborn bdnf+/+ mice. The foliate taste papillae of neonatal bdnf-/- mice had similar defects. We conclude that the normal development of taste neurons requires BDNF.     

CD3- and CD28-dependent induction of PDE7 required for T cell activation

Costimulation of both the CD3 and CD28 receptors is essential for T cell activation. Induction of adenosine 3',5'-monophosphate (cAMP)-specific phosphodiesterase-7 (PDE7) was found to be a consequence of such costimulation. Increased PDE7 in T cells correlated with decreased cAMP, increased interleukin-2 expression, and increased proliferation. Selectively reducing PDE7 expression with a PDE7 antisense oligonucleotide inhibited T cell proliferation; inhibition was reversed by blocking the cAMP signaling pathways that operate through cAMP-dependent protein kinase (PKA). Thus, PDE7 induction and consequent suppression of PKA activity is required for T cell activation, and inhibition of PDE7 could be an approach to treating T cell-dependent disorders.     

Xrcc3 is required for assembly of Rad51 complexes in vivo

Rad51 is a member of a family of eukaryotic proteins related to the bacterial recombinational repair protein RecA. Rad51 protein localizes to multiple subnuclear foci in Chinese hamster ovary cells. Subnuclear Rad51 foci are induced by ionizing radiation or the DNA cross-linking agent cisplatin. Formation of these foci is likely to reflect assembly of a multimeric form of Rad51 that promotes DNA repair. Formation of damage-induced Rad51 foci does not occur in the Chinese hamster ovary cell line irs1SF, which is sensitive to DNA damaging agents. The Rad51 focus formation defect of irs1SF cells is corrected by a construct that encodes the repair protein Xrcc3. Xrcc3 is a human homolog of Rad51 previously isolated by virtue of its ability to correct the radiation sensitivity of irs1SF cells. Changes in the steady state level of Rad51 protein do not account for the irs1SF defect nor do they account for the appearance of foci following DNA damage. These results suggest that Xrcc3 is required for the assembly or stabilization of a multimeric form of Rad51 during DNA repair. Cell lines defective in two different components of DNA protein kinase formed Rad51 foci in response to damage, indicating DNA protein kinase is not required for damaged-induced mobilization of Rad51.     

Tyrosine phosphorylation of cdc2 is required for the replication checkpoint in Schizosaccharomyces pombe

The DNA replication checkpoint inhibits mitosis in cells that are unable to replicate their DNA, as when nucleotide biosynthesis is inhibited by hydroxyurea. In the fission yeast Schizosaccharomyces pombe, genetic evidence suggests that this checkpoint involves the inhibition of Cdc2 activity through the phosphorylation of tyrosine-15. On the contrary, a recent biochemical study indicated that Cdc2 is in an activated state during a replication checkpoint, suggesting that phosphorylation of Cdc2 on tyrosine-15 is not part of the replication checkpoint mechanism. We have undertaken biochemical and genetic studies to resolve this controversy. We report that the DNA replication checkpoint in S. pombe is abrogated in cells that carry the allele cdc2-Y15F, expressing an unphosphorylatable form of Cdc2. Furthermore, Cdc2 isolated from replication checkpoint-arrested cells can be activated in vitro by Cdc25, the tyrosine phosphatase responsible for dephosphorylating Cdc2 in vivo, to the same extent as Cdc2 isolated from cdc25ts-blocked cells, indicating that hydroxyurea treatment causes Cdc2 activity to be maintained at a low level that is insufficient to induce mitosis. These studies show that inhibitory tyrosine-15 phosphorylation of Cdc2 is essential for the DNA replication checkpoint and suggests that Cdc25, and/or one or both of Wee1 and Mik1, the tyrosine kinases that phosphorylate Cdc2, are regulated by the replication checkpoint.     

Identification of phosphorylation sites in the G protein-coupled receptor for parathyroid hormone. Receptor phosphorylation is not required for agonist-induced internalization

In some G protein-coupled receptors (GPCRs), agonist-dependent phosphorylation by specific GPCR kinases (GRKs) is an important mediator of receptor desensitization and endocytosis. Phosphorylation and the subsequent events that it triggers, such as arrestin binding, have been suggested to be regulatory mechanisms for a wide variety of GPCRs. In the present study, we investigated whether agonist-induced phosphorylation of the PTH receptor, a class II GPCR, also regulates receptor internalization. Upon agonist stimulation, the PTH receptor was exclusively phosphorylated on serine residues. Phosphoamino acid analysis of a number of receptor mutants in which individual serine residues had been replaced by threonine identified serine residues in positions 485, 486, and 489 of the cytoplasmic tail as sites of phosphorylation after agonist treatment. When serine residues at positions 483, 485, 486, 489, 495, and 498 were simultaneously replaced by alanine residues, the PTH receptor was no longer phosphorylated either basally or in response to PTH. The substitution of these serine residues by alanine affected neither the number of receptors expressed on the cell surface nor the ability of the receptor to signal via Gs. Overexpression of GRK2, but not GRK3, enhanced PTH-stimulated receptor phosphorylation, and this phosphorylation was abolished by alanine mutagenesis of residues 483, 485, 486, 489, 495, and 498. Thus, phosphorylation of the PTH receptor by the endogenous kinase in HEK-293 cells occurs on the same residues targeted by overexpressed GRK2. Strikingly, the rate and extent of PTH-stimulated internalization of mutated PTH receptors lacking phosphorylation sites were identical to that observed for the wild-type PTH receptor. Moreover, overexpressed GRK2, while enhancing the phosphorylation of the wild-type PTH receptor, had no affect on the rate or extent of receptor internalization in response to PTH. Thus, the agonist-occupied PTH receptor is phosphorylated by a kinase similar or identical to GRK2 in HEK-293 cells, but this phosphorylation is not requisite for efficient receptor endocytosis.     

c-Ras is required for the activation of the matrix metalloproteinases by concanavalin A in 3Y1 cells

Concanavalin A (Con A) is known to trigger augmented secretion and proteolytic activation of the matrix metalloproteinases (MMPs) in fibroblasts. To study the signaling pathway critical for the activation of MMPs in fibroblasts, we examined the effects of dominant negative ras (S17N ras) expression under the control of conditionally inducible promoter in Con A-activated 3Y1 cells. We found that augmented secretion and proteolytic activation of MMP-2 and MMP-9 together with expression of MT1-MMP in Con A-activated 3Y1 were dramatically suppressed by S17N ras expression. These results strongly suggest that c-Ras plays a critical role in the augmented expression and proteolytic activation of MMPs in fibroblasts.     

Synaptotagmin-1 is required for fibroblast growth factor-1 release

By using p65 synaptotagmin-1 and fibroblast growth factor (FGF)-1:beta-galactosidase (beta-gal) NIH 3T3 cell co-transfectants, we demonstrate that a proteolytic fragment consisting of the extravesicular domain of synaptotagmin-1 is released into the extracellular compartment in response to temperature stress with similar kinetics and pharmacological properties as FGF-1:beta-gal. Using a deletion mutant that lacks 95 amino acids from the extravesicular domain of synaptotagmin-1, neither synaptotagmin-1 nor FGF-1:beta-gal are able to access the stress-induced release pathway. Furthermore, the p40 extravesicular fragment of synaptotagmin-1 is constitutively released in p40 synaptotagmin-1 NIH 3T3 cell transfectants, and this release is potentiated when the cells are subjected to temperature stress. These data demonstrate that the p40 fragment derived from synaptotagmin-1 is able to utilize the FGF-1 non-classical exocytotic pathway and that the release of FGF-1 is dependent on synaptotagmin-1.     

The MEK1 proline-rich insert is required for efficient activation of the mitogen-activated protein kinases ERK1 and ERK2 in mammalian cells

MEK1 and MEK2 contain a proline-rich insert not present in any other known MEK (MAP (mitogen-activated protein)/ERK (extracellular signal-regulated kinase) kinase) family members. We examined the effect of removing the MEK1 polyproline insert on MEK activity, its binding to Raf, and its ability to activate ERKs in cells. Deletion of the insert had no effect on either the activity of MEK1 or on its ability to bind to Raf-1. Both wild type and constitutively active MEK1 coimmunoprecipitated with Raf-1 whether or not the insert was present. Deletion of the insert did not reduce activation of MEK1 by EGF or activated Raf in cells. The proline-rich insert enhanced the ability of an otherwise equally active MEK1 protein to regulate endogenous ERKs in mammalian cells. Overexpression of either constitutively active MEK1 lacking the insert or ERK2 compensates for the weaker in vivo activity of the MEK1 deletion mutant. Expression of the insert in cells reduced activation of ERKs by EGF. We conclude that the proline-rich insert is not the site of the MEK-Raf interaction and that the polyproline insert is required for its efficient activation of downstream ERKs in cells.