FAK dimerization controls its kinase-dependent functions at focal adhesions.

Focal adhesion kinase (FAK) controls adhesion-dependent cell motility, survival, and proliferation. FAK has kinase-dependent and kinase-independent functions, both of which play major roles in embryogenesis and tumor invasiveness. The precise mechanisms of FAK activation are not known. Using x-ray crystallography, small angle x-ray scattering, and biochemical and functional analyses, we show that the key step for activation of FAK's kinase-dependent functions--autophosphorylation of tyrosine-397--requires site-specific dimerization of FAK. The dimers form via the association of the N-terminal FERM domain of FAK and are stabilized by an interaction between FERM and the C-terminal FAT domain. FAT binds to a basic motif on FERM that regulates co-activation and nuclear localization. FAK dimerization requires local enrichment, which occurs specifically at focal adhesions. Paxillin plays a dual role, by recruiting FAK to focal adhesions and by reinforcing the FAT:FERM interaction. Our results provide a structural and mechanistic framework to explain how FAK combines multiple stimuli into a site-specific function. The dimer interfaces we describe are promising targets for blocking FAK activation.

Focal adhesion kinase negatively regulates Lck function downstream of the T cell antigen receptor.

Focal adhesion kinase (FAK) is a critical regulator of signal transduction in multiple cell types. Although this protein is activated upon TCR engagement, the cellular function that FAK plays in mature human T cells is unknown. By suppressing the function of FAK, we revealed that FAK inhibits TCR-mediated signaling by recruiting C-terminal Src kinase to the membrane and/or receptor complex following TCR activation. Thus, in the absence of FAK, the inhibitory phosphorylation of Lck and/or Fyn is impaired. Together, these data highlight a novel role for FAK as a negative regulator TCR function in human T cells. These results also suggest that changes in FAK expression could modulate sensitivity to TCR stimulation and contribute to the progression of T cell malignancies and autoimmune diseases.

Caveolin-1 Tyr14 phosphorylation induces interaction with TLR4 in endothelial cells and mediates MyD88-dependent signaling and sepsis-induced lung inflammation.

Activation of TLR4 by the endotoxin LPS is a critical event in the pathogenesis of Gram-negative sepsis. Caveolin-1, the signaling protein associated with caveolae, is implicated in regulating the lung inflammatory response to LPS; however, the mechanism is not understood. In this study, we investigated the role of caveolin-1 in regulating TLR4 signaling in endothelial cells. We observed that LPS interaction with CD14 in endothelial cells induced Src-dependent caveolin-1 phosphorylation at Tyr(14). Using a TLR4-MD2-CD14-transfected HEK-293 cell line and caveolin-1-deficient (cav-1(-/-)) mouse lung microvascular endothelial cells, we demonstrated that caveolin-1 phosphorylation at Tyr(14) following LPS exposure induced caveolin-1 and TLR4 interaction and, thereby, TLR4 activation of MyD88, leading to NF-κB activation and generation of proinflammatory cytokines. Exogenous expression of phosphorylation-deficient Y14F caveolin-1 mutant in cav-1(-/-) mouse pulmonary vasculature rendered the mice resistant to LPS compared with reintroduction of wild-type caveolin-1. Thus, caveolin-1 Y14 phosphorylation was required for the interaction with TLR4 and activation of TLR4-MyD88 signaling and sepsis-induced lung inflammation. Inhibiting caveolin-1 Tyr(14) phosphorylation and resultant inactivation of TLR4 signaling in pulmonary vascular endothelial cells represent a novel strategy for preventing sepsis-induced lung inflammation and injury.

The receptor Ly108 functions as a SAP adaptor-dependent on-off switch for T cell help to B cells and NKT cell development.

Humans and mice deficient in the adaptor protein SAP (Sh2d1a) have a major defect in humoral immunity, resulting from a lack of T cell help for B cells. The role of SAP in this process is incompletely understood. We found that deletion of receptor Ly108 (Slamf6) in CD4(+) T cells reversed the Sh2d1a(-/-) phenotype, eliminating the SAP requirement for germinal centers. This potent negative signaling by Ly108 required immunotyrosine switch motifs (ITSMs) and SHP-1 recruitment, resulting in high amounts of SHP-1 at the T cell:B cell synapse, limiting T cell:B cell adhesion. Ly108-negative signaling was important not only in CD4(+) T cells; we found that NKT cell differentiation was substantially restored in Slamf6(-/-)Sh2d1a(-/-) mice. The ability of SAP to regulate both positive and negative signals in T cells can explain the severity of SAP deficiency and highlights the importance of SAP and SHP-1 competition for Ly108 ITSM binding as a rheostat for the magnitude of T cell help to B cells.

Large-scale phosphotyrosine proteomic profiling of rat renal collecting duct epithelium reveals predominance of proteins involved in cell polarity determination.

Although extensive phosphoproteomic information is available for renal epithelial cells, previous emphasis has been on phosphorylation of serines and threonines with little focus on tyrosine phosphorylation. Here we have carried out large-scale identification of phosphotyrosine sites in pervanadate-treated native inner medullary collecting ducts of rat, with a view towards identification of physiological processes in epithelial cells that are potentially regulated by tyrosine phosphorylation. The method combined antibody-based affinity purification of tyrosine phosphorylated peptides coupled with immobilized metal ion chromatography to enrich tyrosine phosphopeptides, which were identified by LC-MS/MS. A total of 418 unique tyrosine phosphorylation sites in 273 proteins were identified. A large fraction of these sites have not been previously reported on standard phosphoproteomic databases. All results are accessible via an online database: http://helixweb.nih.gov/ESBL/Database/iPY/. Analysis of surrounding sequences revealed four overrepresented motifs: [D/E]xxY*, Y*xxP, DY*, and Y*E, where the asterisk symbol indicates the site of phosphorylation. These motifs plus contextual information, integrated using the NetworKIN tool, suggest that the protein tyrosine kinases involved include members of the insulin- and ephrin-receptor kinase families. Analysis of the gene ontology (GO) terms and KEGG pathways whose protein elements are overrepresented in our data set point to structures involved in epithelial cell-cell and cell-matrix interactions ("adherens junction," "tight junction," and "focal adhesion") and to components of the actin cytoskeleton as major sites of tyrosine phosphorylation in these cells. In general, these findings mesh well with evidence that tyrosine phosphorylation plays a key role in epithelial polarity determination.

The SH2 domain-containing proteins in 21 species establish the provenance and scope of phosphotyrosine signaling in eukaryotes.

The Src homology 2 (SH2) domains are participants in metazoan signal transduction, acting as primary mediators for regulated protein-protein interactions with tyrosine-phosphorylated substrates. Here, we describe the origin and evolution of SH2 domain proteins by means of sequence analysis from 21 eukaryotic organisms from the basal unicellular eukaryotes, where SH2 domains first appeared, through the multicellular animals and increasingly complex metazoans. On the basis of our results, SH2 domains and phosphotyrosine signaling emerged in the early Unikonta, and the numbers of SH2 domains expanded in the choanoflagellate and metazoan lineages with the development of tyrosine kinases, leading to rapid elaboration of phosphotyrosine signaling in early multicellular animals. Our results also indicated that SH2 domains coevolved and the number of the domains expanded alongside protein tyrosine kinases and tyrosine phosphatases, thereby coupling phosphotyrosine signaling to downstream signaling networks. Gene duplication combined with domain gain or loss produced novel SH2-containing proteins that function within phosphotyrosine signaling, which likely have contributed to diversity and complexity in metazoans. We found that intra- and intermolecular interactions within and between SH2 domain proteins increased in prevalence along with organismal complexity and may function to generate more highly connected and robust phosphotyrosine signaling networks.

Focal adhesion kinase promotes integrin adhesion dynamics necessary for chemotropic turning of nerve growth cones.

The ability of extending axons to navigate using combinations of extracellular cues is essential for proper neural network formation. One intracellular signaling molecule that integrates convergent signals from both extracellular matrix (ECM) proteins and growth factors is focal adhesion kinase (FAK). Analysis of FAK function shows that it influences a variety of cellular activities, including cell motility, proliferation, and differentiation. Recent work in developing neurons has shown that FAK and Src function downstream of both attractive and repulsive growth factors, but little is known about the effectors or cellular mechanisms that FAK controls in growth cones on ECM proteins. We report that FAK functions downstream of brain-derived neurotrophic factor (BDNF) and laminin in the modulation of point contact dynamics, phosphotyrosine signaling at filopodial tips, and lamellipodial protrusion. BDNF stimulation accelerates paxillin-containing point contact turnover and formation. Knockdown of FAK function either with a FAK antisense morpholino or by expression of FRNK, a dominant-negative FAK isoform, blocks all aspects of the response to BDNF, including the acceleration of point contact dynamics. On the other hand, expression of specific FAK point mutants can selectively disrupt distinct aspects of the response to BDNF. We also show that growth cone turning depends on both signaling cascades tested here. Finally, we provide the first evidence that growth cone point contacts are asymmetrically regulated during turning to an attractive guidance cue.

Role of glycerol-3-phosphate dehydrogenase 2 in mouse sperm capacitation.

A tyrosine phosphoproteome study of hamster spermatozoa indicated that glycerol-3-phosphate dehydrogenase 2 (GPD2), is one of the proteins that enables tyrosine phosphorylation during sperm capacitation. Further, enzymatic activity of GPD2 correlated positively with sperm capacitation [Kota et al., 2009; Proteomics 9:1809-1826]. Therefore, understanding the function of GPD2 would help to unravel the molecular mechanism of sperm capacitation. In this study, involving the use of spermatozoa from Gpd2(+/+) and Gpd2(-/-) mice, it has been demonstrated that in the absence of Gpd2, hyperactivation and acrosome reaction were significantly altered, and a few changes in protein tyrosine phosphorylation were also observed during capacitation. Evidence is provided to demonstrate that GPD2 activity is required for ROS generation in mouse spermatozoa during capacitation, failing which, capacitation is impaired. These results imply that GPD2 is involved in sperm capacitation.

Function of activation loop tyrosine phosphorylation in the mechanism of c-Kit auto-activation and its implication in sunitinib resistance.

The activation of receptor tyrosine kinases (RTKs) is tightly regulated through a variety of mechanisms. Kinetic studies show that activation of c-Kit RTK occurs through an inter-molecular autophosphorylation. Phosphopeptide mapping of c-Kit reveals that 14-22 phosphates are added to each mol of wild-type (WT) c-Kit during the activation. Phosphorylation sites are found on the JM, kinase insert (KID), c-terminal domains and the activation loop (A-loop), but only the sites on the JM domain contribute to the kinase activation. The A-loop tyrosine (Y(823)) is not phosphorylated until very late in the activation (>90% completion), indicating that the A-loop phosphorylation is not required for c-Kit activation. A sunitinib-resistant mutant D816H that accelerates auto-activation by 184-fold shows no phosphorylation on the A-loop tyrosine after full activation. A loss-of-phosphorylation mutation Y823F remains fully competent in auto-activation. Similar to WT and D816H, the unactivated Y823F mutant binds sunitinib and imatinib with high affinity (K(D) = 5.9 nM). But unlike the WT and D816H where the activated enzymes lose the ability to bind the two drugs, activated Y823F binds the two inhibitors effectively. These observations suggest that the A-loop of activated Y823F remains flexible and can readily adopt unactivated conformations to accommodate DFG-out binders.

Quantitative phosphoproteomics--an emerging key technology in signal-transduction research.

Protein phosphorylation is the most important type of reversible post-translational modification involved in the regulation of cellular signal-transduction processes. In addition to controlling normal cellular physiology on the molecular level, perturbations of phosphorylation-based signaling networks and cascades have been implicated in the onset and progression of various human diseases. Recent advances in mass spectrometry-based proteomics helped to overcome many of the previous limitations in protein phosphorylation analysis. Improved isotope labeling and phosphopeptide enrichment strategies in conjunction with more powerful mass spectrometers and advances in data analysis have been integrated in highly efficient phosphoproteomics workflows, which are capable of monitoring up to several thousands of site-specific phosphorylation events within one large-scale analysis. Combined with ongoing efforts to define kinase-substrate relationships in intact cells, these major achievements have considerable potential to assess phosphorylation-based signaling networks on a system-wide scale. Here, we provide an overview of these exciting developments and their potential to transform signal-transduction research into a technology-driven, high-throughput science.

Phosphorylation of human Jak3 at tyrosines 904 and 939 positively regulates its activity.

Janus tyrosine kinase 3 (Jak3) is essential for signaling by interleukin-2 (IL-2) family cytokines and proper immune function. Dysfunctional regulation of Jak3 may result in certain disease states. However, the molecular mechanisms governing Jak3 activation are not fully understood. In this study, we used a functional-proteomics approach to identify two novel tyrosine phosphorylation sites within Jak3, Y904 and Y939, which are conserved among Jak family proteins. By using phosphospecific antibodies, both residues were observed to be rapidly induced by stimulation of cells with IL-2 or other gammac cytokines. Mechanistic studies indicated that Y904 and Y939 regulate Jak3 activities. A phenylalanine substitution at either site greatly reduced Jak3 kinase activity in vitro and its ability to phosphorylate signal transducer and activator of transcription 5 (Stat5) in vivo, suggesting that phosphorylation of these previously unrecognized residues positively regulates Jak3 activity. Y904 and Y939 were required for optimal ATP usage by Jak3, while phosphorylation of Y939 preferentially promoted Stat5 activity in intact cells. Together, these findings demonstrate positive functional roles for two novel Jak3 phosphoregulatory sites which may be similarly important for other Jak family members. Identification of these sites also provides new therapeutic opportunities to modulate Jak3 function.

The radioprotector O-phospho-tyrosine stimulates DNA-repair via epidermal growth factor receptor- and DNA-dependent kinase phosphorylation.

BACKGROUND AND PURPOSE: Purpose of the study was to elucidate the underlying molecular mechanism of the radioprotector O-phospho-tyrosine (P-Tyr). METHODS: Molecular effects of P-Tyr at the level of EGFR responses were investigated in vitro with bronchial carcinoma cell line A549. Nuclear EGFR transport and DNA-PK activation were quantified after Western blotting. Residual DNA-damages were quantified by help of gammaH(2)AX focus assay. RESULTS: As determined by dose-response curves, treatment of cells with P-Tyr for 16h before irradiation results in radioprotection. Simultaneous treatment with EGFR blocking antibody Cetuximab abolished P-Tyr associated radioprotection. At the molecular level P-Tyr mediated a general phosphorylation of EGFR and a pronounced phosphorylation of nuclear EGFR at residue Thr No. 654, also observed after treatment with ionizing radiation. This phosphorylation was associated with nuclear EGFR accumulation. Moreover, P-Tyr-triggered EGFR nuclear accumulation was associated with phosphorylation of DNA-PK at Thr 2609. This activated form of DNA-PK was not DNA associated, but after radiation, DNA binding increased, particularly after P-Tyr pre-treatment. These molecular effects of P-Tyr resulted in a reduction of residual DNA-damage after irradiation. CONCLUSIONS: Radioprotection by P-Tyr is mediated through its stimulation of nuclear EGFR transport and concurrent, but DNA-damage independent, activation of DNA-PK. Thus, subsequent irradiation results in increased binding of DNA-PK to DNA, improved DNA-repair and increased cell survival.

Relaxin signalling in THP-1 cells uses a novel phosphotyrosine-dependent pathway.

The heterodimeric peptide hormone relaxin acts through the novel G-protein coupled receptor LGR7 to elicit the production of cAMP in the human monocyte cell line THP-1. The very small number of receptors on the cell surface, and the lack of response in cell membranes imply the involvement of a cytoplasmic signal amplification process. Here we show that this process comprises a novel and specific tyrosine kinase activity close to the receptor, and involves neither protein kinase A, mitogen-activated protein kinase, nor phosphoinositide-3 kinase activities as major upstream components. Furthermore, this novel involvement of a tyrosine kinase activity is cell-type dependent, being largely absent from LGR7-transfected HEK293T cells, and receptor-dependent; vasoactive intestinal peptide or isoproterenol signalling in the same cells does not require this tyrosine kinase activity.

Phosphotyrosine signaling in platelets: lessons for vascular thrombosis.

Platelet activation is crucial for normal hemostasis to arrest bleeding following vascular injury. However, excessive platelet activation in narrowed atherosclerotic blood vessels that are subject to high shear forces may initiate the onset of arterial thrombosis. When platelets come into contact with, and adhere to collagen exposed by damaged endothelium, they undergo morphological and functional changes necessary to generate a platelet-rich thrombus. This process is complex and involves precise co-ordination of various signaling pathways which lead to firm platelet adhesion to sites of tissue damage, release of granule contents from activated platelets, platelet shape change, platelet aggregation and subsequent thrombus formation and consolidation. Induction of tyrosine phosphorylation of key signaling molecules has emerged as a critical event central to stimulatory signaling pathways that generate platelet activation, but is an essential component associated with regulatory pathways that limit the extent of platelet activation. Understanding mechanisms that regulate platelet activation may contribute to the development of novel therapeutics that control common vascular diseases such as myocardial infarction and ischaemic stroke.

Effects of HER2 overexpression on cell signaling networks governing proliferation and migration.

Although human epidermal growth factor receptor 2 (HER2) overexpression is implicated in tumor progression for a variety of cancer types, how it dysregulates signaling networks governing cell behavioral functions is poorly understood. To address this problem, we use quantitative mass spectrometry to analyze dynamic effects of HER2 overexpression on phosphotyrosine signaling in human mammary epithelial cells stimulated by epidermal growth factor (EGF) or heregulin (HRG). Data generated from this analysis reveal that EGF stimulation of HER2-overexpressing cells activates multiple signaling pathways to stimulate migration, whereas HRG stimulation of these cells results in amplification of a specific subset of the migration signaling network. Self-organizing map analysis of the phosphoproteomic data set permitted elucidation of network modules differentially regulated in HER2-overexpressing cells in comparison with parental cells for EGF and HRG treatment. Partial least-squares regression analysis of the same data set identified quantitative combinations of signals within the networks that strongly correlate with cell proliferation and migration measured under the same battery of conditions. Combining these modeling approaches enabled association of epidermal growth factor receptor family dimerization to activation of specific phosphorylation sites, which appear to most critically regulate proliferation and/or migration.

The enteropathogenic Escherichia coli EspF effector molecule inhibits PI-3 kinase-mediated uptake independently of mitochondrial targeting.

Delivery of effector molecules into LMme(v) macrophages by enteropathogenic Escherichia coli, via its type three secretion system (T3SS), inhibits bacterial uptake by a phosphatidylinositol-3 (PI-3) kinase-dependent pathway. The T3SS system, encoded by the locus of enterocyte effacement (LEE) pathogenicity island, delivers LEE- and non-LEE-encoded effector proteins into host cells. Previous studies discounted essential roles for the LEE-encoded Map, EspF, Tir or Intimin proteins in this process but correlated it with loss of phosphorylation of the PI-3 kinase substrate, Akt (Celli et al., 2001, EMBO J 20: 1245-1258). Given the more recent finding that these bacterial proteins are multifunctional and can act together to subvert host cellular processes, we generated a quadruple deletion mutant (Map, Tir, EspF and Intimin deficient) to unearth any cooperativity in inhibiting uptake. The quadruple mutant was as defective as the T3SS-defective strain at preventing bacterial uptake with further studies revealing a surprising dependence on EspF but not Map, Tir or Intimin. Subversive activities previously associated with EspF are disruption of epithelial barrier function and programmed cell death, with the latter linked to EspF targeting mitochondria. Interestingly, the C-terminal domain possesses a polyproline motif associated with protein-protein interactions. We demonstrate that EspF-mediated inhibition of PI-3 kinase-dependent uptake: (i) is independent of mitochondrial targeting, (ii) requires the N-terminal domain with and (iii) the C-terminal domain is sufficient to disrupt barrier function but not inhibition of bacterial uptake. Moreover, loss of PI-3 kinase-dependent phosphorylation of Akt and gross changes in host phosphotyrosine protein profiles could not be linked to inhibition of the PI-3 kinase-dependent uptake process.

B and T lymphocyte attenuator-mediated signal transduction provides a potent inhibitory signal to primary human CD4 T cells that can be initiated by multiple phosphotyrosine motifs.

The B and T lymphocyte attenuator (BTLA) is a recently identified member of the CD28 family of cell receptors. Initial reports demonstrated that mice deficient in BTLA expression were more susceptible to experimental autoimmune encephalomyelitis, indicating that BTLA was likely to function as a negative regulator of T cell activation. However, cross-linking of BTLA only resulted in a 2-fold reduction of IL-2 production, questioning the potency with which BTLA engagement blocks T cell activation. We established a model in which BTLA signaling could be studied in primary human CD4 T cells. We observed that cross-linking of a chimeric receptor consisting of the murine CD28 extracellular domain and human BTLA cytoplasmic tail potently inhibits IL-2 production and completely suppresses T cell expansion. Mutation of any BTLA tyrosine motifs had no effect on the ability of BTLA to block T cell activation. Only mutation of all four tyrosines rendered the BTLA cytoplasmic tail nonfunctional. We performed structure-function studies to determine which factors recruited to the BTLA cytoplasmic tail correlated with BTLA function. Using pervanadate as a means to phosphorylate the BTLA cytoplasmic tail, we observed both Src homology protein (SHP)-1 and SHP-2 recruitment. However, upon receptor engagement, we observed only SHP-1 recruitment, and mutations that abrogated SHP-1 recruitment did not impair BTLA function. These studies question whether SHP-1 or SHP-2 have any role in BTLA function and caution against the use of pervanadate as means to initiate signal transduction cascades in primary cells.

Signals for stress erythropoiesis are integrated via an erythropoietin receptor-phosphotyrosine-343-Stat5 axis.

Anemia due to chronic disease or chemotherapy often is ameliorated by erythropoietin (Epo). Present studies reveal that, unlike steady-state erythropoiesis, erythropoiesis during anemia depends sharply on an Epo receptor-phosphotyrosine-343-Stat5 signaling axis. In mice expressing a phosphotyrosine-null (PY-null) Epo receptor allele (EpoR-HM), severe and persistent anemia was induced by hemolysis or 5-fluorouracil. In short-term transplantation experiments, donor EpoR-HM bone marrow cells also failed to efficiently repopulate the erythroid compartment. In each context, stress erythropoiesis was rescued to WT levels upon the selective restoration of an EpoR PY343 Stat5-binding site (EpoR-H allele). As studied using a unique primary culture system, EpoR-HM erythroblasts exhibited marked stage-specific losses in Epo-dependent growth and survival. EpoR-H PY343 signals restored efficient erythroblast expansion, and the selective Epo induction of the Stat5 target genes proviral integration site-1 (Pim-1) and oncostatin-M. Bcl2-like 1 (Bcl-x), in contrast, was not significantly induced via WT-EpoR, EpoR-HM, or EpoR-H alleles. In Kit+ CD71+ erythroblasts, EpoR-PY343 signals furthermore enhanced SCF growth effects, and SCF modulation of Pim-1 kinase and oncostatin-M expression. In maturing Kit- CD71+ erythroblasts, oncostatin-M exerted antiapoptotic effects that likewise depended on EpoR PY343-mediated events. Stress erythropoiesis, therefore, requires stage-specific EpoR-PY343-Stat5 signals, some of which selectively bolster SCF and oncostatin-M action.

Quantitative phosphotyrosine proteomics of EphB2 signaling by stable isotope labeling with amino acids in cell culture (SILAC).

Eph-related receptor tyrosine kinases (RTK) have been implicated in several biological functions including synaptic plasticity, axon guidance, and morphogenesis, yet the details of the signal transduction pathways that produce these specific biological functions after ligand-receptor interaction remain unclear. We used Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) in combination with LC-MS/MS to characterize cellular signaling following stimulation by ephrinB1-Fc of NG-108 cells that overexpress EphB2 receptors. Because tyrosine phosphorylation functions as a key regulatory event in RTK signaling, we used anti-phosphotyrosine immunoprecipitation (pY IP) of cell lysates to isolate potential participants in the EphB2 pathway. Our SILAC experiments identified 127 unique proteins, 40 of which demonstrated increased abundance in pY IPs from ephrinB1-Fc stimulated cells as compared with unstimulated cells. Six proteins demonstrated decreased abundance, and 81 did not change significantly in relative abundance. Western blotting analysis of five proteins after pY IP verified their SILAC results. On the basis of previously published work and use of PathwayAssist software, we proposed an interaction network downstream of EphB2 for the proteins with changed ratios.