Tyrosine Phosphorylation of Integrin β3 Regulates Kindlin-2 Binding and Integrin Activation

Kindlins are essential for integrin activation in cell systems and do so by working in a cooperative fashion with talin via their direct interaction with integrin β cytoplasmic tails (CTs). Kindlins interact with the membrane-distal NxxY motif, which is distinct from the talin-binding site within the membrane-proximal NxxY motif. The Tyr residues in both motifs can be phosphorylated, and it has been suggested that this modification of the membrane-proximal NxxY motif negatively regulates interaction with the talin head domain. However, the influence of Tyr phosphorylation of the membrane-distal NxxY motif on kindlin binding is unknown. Using mutational analyses and phosphorylated peptides, we show that phosphorylation of the membrane-distal NITY⁷⁵⁹ motif in the β₃ CT disrupts kindlin-2 recognition. Phosphorylation of this membrane-distal Tyr also disables the ability of kindlin-2 to coactivate the integrin. In direct binding studies, peptides corresponding to the non-phosphorylated β₃ CT interacted well with kindlin-2, whereas the Tyr⁷⁵⁹-phosphorylated peptide failed to bind kindlin-2 with measurable affinity. These observations indicate that transitions between the phosphorylated and non-phosphorylated states of the integrin β₃ CT determine reactivity with kindlin-2 and govern the role of kindlin-2 in regulating integrin activation.     

�� Integrin Tyrosine Phosphorylation Is a Conserved Mechanism for Regulating Talin-induced Integrin Activation

Integrins are large membrane-spanning receptors fundamental to cell adhesion and migration. Integrin adhesiveness for the extracellular matrix is activated by the cytoskeletal protein talin via direct binding of its phosphotyrosine-binding-like F3 domain to the cytoplasmic tail of the β integrin subunit. The phosphotyrosine-binding domain of the signaling protein Dok1, on the other hand, has an inactivating effect on integrins, a phenomenon that is modulated by integrin tyrosine phosphorylation. Using full-length tyrosine-phosphorylated ¹⁵N-labeled β3, β1A, and β7 integrin tails and an NMR-based protein-protein interaction assay, we show that talin1 binds to the NPXY motif and the membrane-proximal portion of β3, β1A, and β7 tails, and that the affinity of this interaction is decreased by integrin tyrosine phosphorylation. Dok1 only interacts weakly with unphosphorylated tails, but its affinity is greatly increased by integrin tyrosine phosphorylation. The Dok1 interaction remains restricted to the integrin NPXY region, thus phosphorylation inhibits integrin activation by increasing the affinity of β integrin tails for a talin competitor that does not form activating membrane-proximal interactions with the integrin. Key residues governing these specificities were identified by detailed structural analysis, and talin1 was engineered to bind preferentially to phosphorylated integrins by introducing the mutation D372R. As predicted, this mutation affects talin1 localization in live cells in an integrin phosphorylation-specific manner. Together, these results indicate that tyrosine phosphorylation is a common mechanism for regulating integrin activation, despite subtle differences in how these integrins interact with their binding proteins.     

δ-Opioid Receptor Activation and MicroRNA Expression of the Rat Cortex in Hypoxia

Prolonged hypoxic/ischemic stress may cause cortical injury and clinically manifest as a neurological disability. Activation of the δ-opioid receptor (DOR) may induce cortical protection against hypoxic/ischemic insults. However, the mechanisms underlying DOR protection are not clearly understood. We have recently found that DOR activation modulates the expression of microRNAs (miRNAs) in the kidney exposed to hypoxia, suggesting that DOR protection may involve a miRNA mechanism. To determine if the miRNAs expressed in the cortex mediated DOR neuroprotection, we examined 19 miRNAs that were previously identified as hypoxia- and DOR-regulated miRNAs in the kidney, in the rat cortex treated with UFP-512, a potent and specific DOR agonist under hypoxic condition. Of the 19 miRNAs tested, 17 were significantly altered by hypoxia and/or DOR activation with the direction and amplitude varying depending on hypoxic duration and times of DOR treatment. Expression of several miRNAs such as miR-29b, -101b, -298, 324-3p, -347 and 466b was significantly depressed after 24 hours of hypoxia. Similar changes were seen in normoxic condition 24 hours after DOR activation with one-time treatment of UFP-512. In contrast, some miRNAs were more tolerant to hypoxic stress and showed significant reduction only with 5-day (e.g., miR-31 and -186) or 10-day (e.g., miR-29a, let-7f and -511) exposures. In addition, these miRNAs had differential responses to DOR activation. Other miRNAs like miRs-363* and -370 responded only to the combined exposure to hypoxia and DOR treatment, with a notable reduction of >70% in the 5-day group. These data suggest that cortical miRNAs are highly yet differentially sensitive to hypoxia. DOR activation can modify, enhance or resolve the changes in miRNAs that target HIF, ion transport, axonal guidance, free radical signaling, apoptosis and many other functions.     

Regulation of autophagy by p38α MAPK

Autophagy is induced in mammalian cells by nutrient deprivation, which acts through repression of the protein kinase mammalian target of rapamycin (mTOR) and may involve other unknown mechanisms. Mitogen-activated protein kinases (MAPKs), and in particular p38 MAPK, are implicated in amino acid signalling. Furthermore, the extracellular signal-regulated kinase (ERK) and p38 regulate autophagy in response to various stimuli. However, the molecular mechanisms of p38 regulation of autophagy are still widely unknown. Our recent data suggest that p38α MAPK negatively regulates the interaction of mAtg9 and a novel mAtg9 binding partner, p38IP, to control the levels of autophagy induced in response to starvation.     

Activation of Toll-Like Receptor 2 Prevents Suppression of T-Cell Interferon γ Production by Modulating p38/Extracellular Signal-Regulated Kinase Pathways following Alcohol and Burn Injury

Recent studies indicate that toll-like receptors (TLRs) are expressed on T cells and that these receptors directly or indirectly activate the adaptive immune system. We have shown previously that acute alcohol/ethanol (EtOH) intoxication combined with burn injury suppresses mesenteric lymph node (MLN) T-cell interleukin-2 (IL-2) and interferon γ (IFN-γ) production. We examined whether direct stimulation of T cells with TLR2, 4, 5 and 7 agonists modulates CD3-mediated T-cell IL-2/IFN-γ release following EtOH and burn injury. Male mice were gavaged with EtOH (2.9 gm/kg) 4 h prior to receiving an ~12.5% total body surface area sham or full-thickness burn injury. Animals were killed on d 1 after injury and T cells were purified from MLN and spleens. T cells were cultured with plate-bound anti-CD3 in the presence or absence of various TLR ligands. Although TLR2, 4 and 5 agonists potentiate anti-CD3–dependent IFN-γ by T cells, the TLR2 agonist alone induced IFN-γ production independent of CD3 stimulation. Furthermore, T cells were treated with inhibitors of myeloid differentiation primary response protein 88 (MyD88), TIR domain-containing adaptor protein (TIRAP), p38 and/or extracellular signal-regulated kinase (ERK) to determine the mechanism by which TLR2 mediates IL-2/IFN-γ production. IL-2 was not influenced by TLR agonists. MyD88 and TIRAP inhibitory peptides dose-dependently diminished the ability of T cells to release IFN-γ. p38 and ERK inhibitors also abolished TLR2-mediated T-cell IFN-γ. Together, our findings suggest that TLR2 directly modulates T-cell IFN-γ production following EtOH and burn injury, independent of antigen-presenting cells. Furthermore, we demonstrated that MyD88/TIRAP-dependent p38/ERK activation is critical to TLR2-mediated T-cell IFN-γ release following EtOH and burn injury.     

Activation of Toll-Like Receptor 2 Prevents Suppression of T-Cell Interferon γ Production by Modulating p38/Extracellular Signal-Regulated Kinase Pathways following Alcohol and Burn Injury

Recent studies indicate that toll-like receptors (TLRs) are expressed on T cells and that these receptors directly or indirectly activate the adaptive immune system. We have shown previously that acute alcohol/ethanol (EtOH) intoxication combined with burn injury suppresses mesenteric lymph node (MLN) T-cell interleukin-2 (IL-2) and interferon γ (IFN-γ) production. We examined whether direct stimulation of T cells with TLR2, 4, 5 and 7 agonists modulates CD3-mediated T-cell IL-2/IFN-γ release following EtOH and burn injury. Male mice were gavaged with EtOH (2.9 gm/kg) 4 h prior to receiving an ~12.5% total body surface area sham or full-thickness burn injury. Animals were killed on d 1 after injury and T cells were purified from MLN and spleens. T cells were cultured with plate-bound anti-CD3 in the presence or absence of various TLR ligands. Although TLR2, 4 and 5 agonists potentiate anti-CD3-dependent IFN-γ by T cells, the TLR2 agonist alone induced IFN-γ production independent of CD3 stimulation. Furthermore, T cells were treated with inhibitors of myeloid differentiation primary response protein 88 (MyD88), TIR domain-containing adaptor protein (TIRAP), p38 and/or extracellular signal-regulated kinase (ERK) to determine the mechanism by which TLR2 mediates IL-2/IFN-γ production. IL-2 was not influenced by TLR agonists. MyD88 and TIRAP inhibitory peptides dose-dependently diminished the ability of T cells to release IFN-γ. p38 and ERK inhibitors also abolished TLR2-mediated T-cell IFN-γ. Together, our findings suggest that TLR2 directly modulates T-cell IFN-γ production following EtOH and burn injury, independent of antigen-presenting cells. Furthermore, we demonstrated that MyD88/TIRAP-dependent p38/ERK activation is critical to TLR2-mediated T-cell IFN-γ release following EtOH and burn injury.     

Serine Dephosphorylation of Receptor Protein Tyrosine Phosphatase α in Mitosis Induces Src Binding and Activation

Receptor protein tyrosine phosphatase α (RPTPα) is the mitotic activator of the protein tyrosine kinase Src. RPTPα serine hyperphosphorylation was proposed to mediate mitotic activation of Src. We raised phosphospecific antibodies to the two main serine phosphorylation sites, and we discovered that RPTPα Ser204 was almost completely dephosphorylated in mitotic NIH 3T3 and HeLa cells, whereas Ser180 and Tyr789 phosphorylation were only marginally reduced in mitosis. Concomitantly, Src pTyr527 and pTyr416 were dephosphorylated, resulting in 2.3-fold activation of Src in mitosis. Using inhibitors and knockdown experiments, we demonstrated that dephosphorylation of RPTPα pSer204 in mitosis was mediated by PP2A. Mutation of Ser204 to Ala did not activate RPTPα, and intrinsic catalytic activity of RPTPα was not affected in mitosis. Interestingly, binding of endogenous Src to RPTPα was induced in mitosis. GRB2 binding to RPTPα, which was proposed to compete with Src binding to RPTPα, was only modestly reduced in mitosis, which could not account for enhanced Src binding. Moreover, we demonstrate that Src bound to mutant RPTPα-Y789F, lacking the GRB2 binding site, and mutant Src with an impaired Src homology 2 (SH2) domain bound to RPTPα, illustrating that Src binding to RPTPα is not mediated by a pTyr-SH2 interaction. Mutation of RPTPα Ser204 to Asp, mimicking phosphorylation, reduced coimmunoprecipitation with Src, suggesting that phosphorylation of Ser204 prohibits binding to Src. Based on our results, we propose a new model for mitotic activation of Src in which PP2A-mediated dephosphorylation of RPTPα pSer204 facilitates Src binding, leading to RPTPα-mediated dephosphorylation of Src pTyr527 and pTyr416 and hence modest activation of Src.Protein tyrosine phosphatases (PTPs) are responsible for dephosphorylation of the phosphotyrosyl residues. The human genome contains approximately 100 genes that encode members of the four PTP families, and most of them have mouse orthologues (2, 48). According to their subcellular localization, the classical PTPs, encoded by less than half of the total PTP genes, are divided into two subfamilies: cytoplasmic and receptor protein tyrosine phosphatases (RPTPs). The majority of the RPTPs contain, besides a variable extracellular domain and a transmembrane domain, two highly homologous phosphatase domains (27), with the membrane-proximal domain comprising most of the catalytic activity (33).RPTPα is a typical RPTP with a small, highly glycosylated extracellular domain (13). RPTPα function is regulated by many mechanisms, including proteolysis (18), oxidation (55), dimerization (7, 23, 24, 47, 52), and phosphorylation of serine and tyrosine residues (16, 17, 49). RPTPα is broadly expressed in many cell types, and over the years, RPTPα has been shown to be involved in a number of signaling mechanisms, including neuronal (15) and skeletal muscle (34) cell differentiation, neurite elongation (8, 9, 56), insulin receptor signaling downregulation (3, 28, 30, 31, 35), insulin secretion (25), activation of voltage-gated potassium channel Kv1.2 (51), long-term potentiation in hippocampal neurons (32, 38), matrix-dependent force transduction (53), and cell spreading and migration (21, 45, 57).The majority of the roles played in these cellular processes involve RPTPα''s ability to activate the proto-oncogenes Src and Fyn by dephosphorylating their C-terminal inhibitory phosphotyrosine (5, 15, 39, 45, 61). Normally, this phosphotyrosine (pTyr527 in chicken Src) binds to the Src homology 2 (SH2) domain, keeping the protein in an inactive closed conformation. A displacement mechanism was proposed for RPTPα-mediated Src activation in which pTyr789 of RPTPα is required to bind the SH2 domain of Src before RPTPα dephosphorylates Tyr527 (58). This model is the subject of debate since other studies show that RPTPα lacking Tyr789 is still able to dephosphorylate and activate Src (12, 26, 29, 56). In normal cells, Src reaches its activation peak during mitosis (4, 11, 40, 42), and with the help of overexpressing cells, it was shown that this activation is triggered mainly by RPTPα. The model that emerged is that RPTPα is activated in mitosis due to serine hyperphosphorylation and detaches from the GRB2 scaffolding protein (59, 60) that normally binds most of the pTyr789 of RPTPα via its SH2 domain (14, 17, 46). Two serine phosphorylation sites were mapped in the juxtamembrane domain of RPTPα, Ser180 and Ser204 (49). The kinases that were found responsible for their phosphorylation were protein kinase C delta (PKCdelta) (10) and CaMKIIalpha (9), but there is no clear evidence that these kinases are activated in mitosis. We set out to investigate the role of serine phosphorylation of RPTPα in mitotic activation of Src.We generated phosphospecific antibodies and show that RPTPα pSer204, but not pSer180, is dephosphorylated in mitotic NIH 3T3 and HeLa cells, concomitantly with activation of Src. Selective inhibitors suggested that PP2A was the phosphatase that dephosphorylated pSer204. RNA interference (RNAi)-mediated knockdown of the catalytic subunit of PP2A demonstrated that indeed PP2A was responsible for mitotic dephosphorylation of RPTPα pSer204. It is noteworthy that PP2A is known to be activated in mitosis. Intrinsic PTP activities of RPTPα were similar in unsynchronized and mitotic cells, and mutation of Ser204 did not activate RPTPα in in vitro PTP assays. Yet, Src binding to RPTPα was induced in mitotic NIH 3T3 cells and RPTPα-S204D with a phosphomimicking mutation at Ser204 coimmunoprecipitated less efficiently with Src. Based on our results, we propose a mechanism for mitotic activation of Src that is triggered by dephosphorylation of RPTPα pSer204, resulting in enhanced affinity for Src and subsequent dephosphorylation and activation of Src.     

Peptide EphB2/CTF2 Generated by the ��-Secretase Processing of EphB2 Receptor Promotes Tyrosine Phosphorylation and Cell Surface Localization of N-Methyl-d-aspartate Receptors

Presenilin 1, a protein involved in the development of familial Alzheimer disease, is an important functional component of the γ-secretase complex that processes many cell surface receptors including the EphB2 tyrosine kinase receptors (Litterst, C., Georgakopoulos, A., Shioi, J., Ghersi, E., Wisniewski, T., Wang, R., Ludwig, A., and Robakis, N. K. (2007) J. Biol. Chem. 282, 16155–16163). Recent evidence reveals that cytosolic peptides produced by the combined metalloproteinase/γ-secretase processing of cell surface proteins function in signal transduction and protein phosphorylation. Here we show that peptide EphB2/CTF2 released to the cytosol by the γ-secretase processing of EphB2 receptor, has tyrosine kinase activity, and directly phosphorylates the N-methyl-d-aspartate receptor (NMDAR) subunits in both cell lines and primary neuronal cultures. This phosphorylation occurs in the absence of Src kinases and is resistant to Src inhibitors revealing a novel pathway of NMDAR tyrosine phosphorylation independent of Src activity. EphB2/CTF2, but not a kinase-deficient mutant of EphB2/CTF2, promotes the cell surface expression of NMDAR. Because NMDAR plays central roles in synaptic plasticity and function, our results provide a potential link between the γ-secretase function of presenilin 1 and learning and memory.N-Methyl-d-aspartate receptor (NMDAR)⁵ is a subtype of ionotropic glutamate receptors expressed in the mammalian central nervous system. Because of its high calcium permeability, this receptor plays crucial roles in the development of the central nervous system, neuroplasticity, and excitotoxicity. In the adult forebrain NMDAR consists mainly of NR1, NR2A, and NR2B subunits, and its functions are regulated by tyrosine phosphorylation events in these subunits. For example, up-regulation of NMDAR channel functions following subunit phosphorylation by the Src family of nonreceptor tyrosine kinases plays crucial roles in cellular signaling, learning, and memory. In addition, tyrosine phosphorylation of NMDAR is important in pathological conditions including stroke, epilepsy, and chronic pain (for review see Ref. 2).Eph receptors constitute the largest family of transmembrane receptor tyrosine kinases. This family is divided into EphA and EphB subclasses based on their ability to be activated by ephrinA and ephrinB ligands, respectively. EphB-ephrinB interactions have been implicated in brain ontogenesis, repulsive axonal guidance, and dendritic spine morphogenesis (reviewed in Refs. 3 and 4). Mounting evidence suggests that interactions between EphB receptors and NMDARs regulate synapse formation, maturation, and plasticity. For example, activation of EphB2 receptor (EphB2R) by ephrinB ligand leads to the formation of complexes between EphB2R and NMDAR, resulting in increased synaptogenesis (5). In contrast, EphB2R knock-out mice show reduced current activity of synaptic NMDAR and a decreased number of glutamate receptors in excitatory synapses (6, 7). Synaptic plasticity, e.g. long term potentiation and depression, is also impaired in these mice (6, 8). Other evidence suggests that the NR2B subunit is tyrosine-phosphorylated by Src kinases in response to EphB2R activation (9, 10).PS1, a protein involved in the development of familial Alzheimer disease (FAD), is expressed in embryonic and adult brains where it is enriched in neurons (11, 12). Previous work revealed that PS1 is a necessary component of the proteolytic γ-secretase complex that promotes production of the Aβ peptides of Alzheimer disease amyloid by processing APP at the γ-cleavage sites (13, 14). Recent evidence, however, revealed that in addition to APP, the PS/γ-secretase system facilitates the proteolytic processing and signaling of many cell surface transmembrane proteins. Almost all γ-secretase substrates are first processed through a pathway that involves extracellular cleavages, usually by a metalloproteinase (MP), and shedding of their ectodomain, whereas the remaining membrane-associated fragments are cleaved at the epsilon site (ϵ-site) by the PS1/γ-secretase system to produce cytosolic peptides containing the cytoplasmic sequence of the receptor. Many of these peptides have important signal transduction properties including regulation of gene expression and protein phosphorylation (for review see Ref. 15).Recently we reported that EphB2R is processed by the MP/γ-secretase system (1). This processing involves cleavage of the EphB2R extracellular domain close to the transmembrane sequence by MP ADAM10 (a disintegrin and metalloproteinase 10) to produce EphB2/N-terminal fragment that is released to the extracellular medium. The remaining membrane-bound C-terminal fragment, termed EphB2/CTF1, is further processed by the PS/γ-secretase system at the ϵ-site to release cytosolic peptide EphB2/CTF2 containing the cytoplasmic sequence of the receptor where its kinase activity resides (1). We reported recently that processing of EphB2R and production of peptide EphB2/CTF2 is stimulated by ephrinB ligands. In addition, in agreement with recent reports that FAD mutations inhibit the γ-secretase cleavage at the ϵ-site of many substrates (16, 17), we showed that PS1 FAD mutations inhibit production of EphB2/CTF2 (1). Here we report that EphB2/CTF2 has tyrosine kinase activity, phosphorylates the NMDAR subunits in the absence of Src activity, and promotes their surface localization.     

Activation of multiple MAPK pathways (ERKs,JNKs, p38‐MAPK) by diverse stimuli in the amphibian heart

We investigated the expression and activation of three MAPK subfamilies in the isolated perfused amphibian heart. ERK was detected as a 43 kDa band; p38MAPK was detected as a band corresponding to 38 kDa and JNKs were detected as two bands corresponding to 46 and 52 kDa, respectively. PMA induced the activation of the ERK pathway as assessed by determining the phosphorylation state of ERK and the upstream component MEK1/2. PD98059 abolished this activation. p38MAPK was phosphorylated by sorbitol (almost 12fold, maximal within 10–15 min) and JNKs were phosphorylated and activated by sorbitol or anoxia/reoxygenation (approximately 4 and 2.5fold, respectively). SB203580 completely blocked the activation of p38MAPK by sorbitol. These results indicate that the MAPK pathways activated by phorbol esters, hyperosmotic stress or anoxia/reoxygenation in the amphibian heart may have an important role in this experimental system.