Tyrosine Phosphorylation at a Site Highly Conserved in the L1 Family of Cell Adhesion Molecules Abolishes Ankyrin Binding and Increases Lateral Mobility of Neurofascin

This paper presents evidence that a member of the L1 family of ankyrin-binding cell adhesion molecules is a substrate for protein tyrosine kinase(s) and phosphatase(s), identifies the highly conserved FIGQY tyrosine in the cytoplasmic domain as the principal site of phosphorylation, and demonstrates that phosphorylation of the FIGQY tyrosine abolishes ankyrin-binding activity. Neurofascin expressed in neuroblastoma cells is subject to tyrosine phosphorylation after activation of tyrosine kinases by NGF or bFGF or inactivation of tyrosine phosphatases with vanadate or dephostatin. Furthermore, both neurofascin and the related molecule Nr-CAM are tyrosine phosphorylated in a developmentally regulated pattern in rat brain. The FIGQY sequence is present in the cytoplasmic domains of all members of the L1 family of neural cell adhesion molecules. Phosphorylation of the FIGQY tyrosine abolishes ankyrin binding, as determined by coimmunoprecipitation of endogenous ankyrin and in vitro ankyrin-binding assays. Measurements of fluorescence recovery after photobleaching demonstrate that phosphorylation of the FIGQY tyrosine also increases lateral mobility of neurofascin expressed in neuroblastoma cells to the same extent as removal of the cytoplasmic domain. Ankyrin binding, therefore, appears to regulate the dynamic behavior of neurofascin and is the target for regulation by tyrosine phosphorylation in response to external signals. These findings suggest that tyrosine phosphorylation at the FIGQY site represents a highly conserved mechanism, used by the entire class of L1-related cell adhesion molecules, for regulation of ankyrin-dependent connections to the spectrin skeleton.Vertebrate L1, neurofascin, neuroglial cell adhesion molecule (Ng-CAM),¹ Ng-CAM–related cell adhesion molecule (Nr-CAM), and Drosophila neuroglian are members of a family of nervous system cell adhesion molecules that possess variable extracellular domains comprised of Ig and fibronectin type III domains and a relatively conserved cytoplasmic domain (Grumet, 1991; Hortsch and Goodman, 1991; Rathgen and Jessel, 1991; Sonderegger and Rathgen, 1992; Hortsch, 1996). Members of this family, including a number of alternatively spliced forms, are abundant in the nervous system during early development as well as in adults. Neurofascin and Nr-CAM, for example, constitute ∼0.5% of the total membrane protein in adult brain (Davis et al., 1993; Davis and Bennett, 1994). Cellular functions attributed to the L1 family include axon fasciculation (Stallcup and Beasley, 1985; Landmesser et al., 1988; Brummendorf and Rathjen, 1993; Bastmeyer et al., 1995; Itoh et al., 1995; Magyar-Lehmann et al., 1995), axonal guidance (van den Pol and Kim, 1993; Liljelund et al., 1994; Brittis and Silver, 1995; Brittis et al., 1995; Lochter et al., 1995; Wong et al., 1996), neurite extension (Chang et al., 1987; Felsenfeld et al., 1994; Hankin and Lagenaur, 1994; Ignelzi et al., 1994; Williams et al., 1994a ,b,c,d; Doherty et al., 1995; Zhao and Siu, 1995), a role in long term potentiation (Luthl et al., 1994), synaptogenesis (Itoh et al., 1995), and myelination (Wood et al., 1990). The potential clinical importance of this group of proteins has been emphasized by the findings that mutations in the L1 gene on the X chromosome are responsible for developmental anomalies including hydrocephalus and mental retardation (Rosenthal et al., 1992; Jouet et al., 1994; Wong et al., 1995).The conserved cytoplasmic domains of L1 family members include a binding site for the membrane skeletal protein ankyrin. This interaction was first described for neurofascin (Davis et. al., 1993) and subsequently has been observed for L1, Nr-CAM (Davis and Bennett, 1994), and Drosophila neuroglian (Dubreuil et al., 1996). The membrane-binding domain of ankyrin contains two distinct sites for neurofascin and has the potential to promote lateral association of neurofascin and presumably other L1 family members (Michaely and Bennett, 1995). Nodes of Ranvier are physiologically relevant axonal sites where ankyrin and L1 family members collaborate, based on findings of colocalization of a specialized isoform of ankyrin with alternatively spliced forms of neurofascin and NrCAM in adults (Davis et al., 1996) as well as in early axonal developmental intermediates (Lambert, S., J. Davis, P. Michael, and V. Bennett. 1995. Mol. Biol. Cell. 6:98a).L1, after homophilic and/or heterophilic binding, participates in signal transduction pathways that ultimately are associated with neurite extension and outgrowth (Ignelzi et al., 1994; Williams et al., 1994a ,b,c,d; Doherty et al., 1995). L1 copurifies with a serine–threonine protein kinase (Sadoul et al., 1989) and is phosphorylated on a serine residue that is not conserved among other family members (Wong et al., 1996). L1 pathway(s) may also involve G proteins, calcium channels, and tyrosine phosphorylation (Williams et al., 1994a ,b,c,d; Doherty et al., 1995). After homophilic interactions, L1 directly activates a tyrosine signaling cascade after a lateral association of its ectodomain with the fibroblast growth factor receptor (Doherty et al., 1995). Antibodies against L1 have also been shown to activate protein tyrosine phosphatase activity in growth cones (Klinz et al., 1995). However, details of the downstream substrates of L1-promoted phosphorylation and dephosphorylation and possible roles of the cytoplasmic domain are not known.Tyrosine phosphorylation is well established to modulate cell–cell and cell–extracellular matrix interactions involving integrins and their associated proteins (Akiyama et al., 1994; Arroyo et al., 1994; Schlaepfer et al., 1994; Law et al., 1996) as well as the cadherins (Balsamo et al., 1996; Krypta et al., 1996; Brady-Kalnay et al., 1995; Shibamoto et al., 1995; Hoschuetzky et al., 1994; Matsuyoshi et al., 1992). For example, the adhesive functions of the calciumdependent cadherin cell adhesion molecule are mediated by a dynamic balance between tyrosine phosphorylation of β-catenin by TrkA and dephosphorylation via the LARtype protein tyrosine phosphatase (Krypta et al., 1996). In this example the regulation of binding among the structural proteins is the result of a coordination between classes of protein kinases and protein phosphatases.This study presents evidence that neurofascin, expressed in a rat neuroblastoma cell line, is a substrate for both tyrosine kinases and protein tyrosine phosphatases at a tyrosine residue conserved among all members of the L1 family. Site-specific tyrosine phosphorylation promoted by both tyrosine kinase activators (NGF and bFGF) and protein tyrosine phosphatase inhibitors (dephostatin and vanadate) is a strong negative regulator of the neurofascin– ankyrin binding interaction and modulates the membrane dynamic behavior of neurofascin. Furthermore, neurofascin and, to a lesser extent Nr-CAM, are also shown here to be tyrosine phosphorylated in developing rat brain, implying a physiological relevance to this phenomenon. These results indicate that neurofascin may be a target for the coordinate control over phosphorylation that is elicited by protein kinases and phosphatases during in vivo tyrosine phosphorylation cascades. The consequent decrease in ankyrin-binding capacity due to phosphorylation of neurofascin could represent a general mechanism among the L1 family members for regulation of membrane–cytoskeletal interactions in both developing and adult nervous systems.     

Semaphorin3A Signaling Mediated by Fyn-dependent Tyrosine Phosphorylation of Collapsin Response Mediator Protein 2 at Tyrosine 32

Collapsin response mediator protein 2 (CRMP2) is an intracellular protein that mediates signaling of Semaphorin3A (Sema3A), a repulsive axon guidance molecule. Fyn, a Src-type tyrosine kinase, is involved in the Sema3A signaling. However, the relationship between CRMP2 and Fyn in this signaling pathway is still unknown. In our research, we demonstrated that Fyn phosphorylated CRMP2 at Tyr³² residues in HEK293T cells. Immunohistochemical analysis using a phospho-specific antibody at Tyr³² of CRMP showed that Tyr³²-phosphorylated CRMP was abundant in the nervous system, including dorsal root ganglion neurons, the molecular and Purkinje cell layer of adult cerebellum, and hippocampal fimbria. Overexpression of a nonphosphorylated mutant (Tyr³² to Phe³²) of CRMP2 in dorsal root ganglion neurons interfered with Sema3A-induced growth cone collapse response. These results suggest that Fyn-dependent phosphorylation of CRMP2 at Tyr³² is involved in Sema3A signaling.Collapsin response mediator proteins (CRMPs)⁴ have been identified as intracellular proteins that mediate Semaphorin3A (Sema3A) signaling in the nervous system (1). CRMP2 is one of the five members of the CRMP family. CRMPs also mediate signal transduction of NT3, Ephrin, and Reelin (2–4). CRMPs interact with several intracellular molecules, including tubulin, Numb, kinesin1, and Sra1 (5–8). CRMPs are involved in axon guidance, axonal elongation, cell migration, synapse maturation, and the generation of neuronal polarity (1, 2, 4, 5).CRMP family proteins are known to be the major phosphoproteins in the developing brain (1, 9). CRMP2 is phosphorylated by several Ser/Thr kinases, such as Rho kinase, cyclin-dependent kinase 5 (Cdk5), and glycogen synthase kinase 3β (GSK3β) (2, 10–13). The phosphorylation sites of CRMP2 by these kinases are clustered in the C terminus and have already been identified. Rho kinase phosphorylates CRMP2 at Thr⁵⁵⁵ (10). Cdk5 phosphorylates CRMP2 at Ser⁵²², and this phosphorylation is essential for sequential phosphorylations by GSK3β at Ser⁵¹⁸, Thr⁵¹⁴, and Thr⁵⁰⁹ (2, 11–13). These phosphorylations disrupt the interaction of CRMP2 with tubulin or Numb (2, 3, 13). The sequential phosphorylation of CRMP2 by Cdk5 and GSK3β is an essential step in Sema3A signaling (11, 13). Furthermore, the neurofibrillary tangles in the brains of people with Alzheimer disease contain hyperphosphorylated CRMP2 at Thr⁵⁰⁹, Ser⁵¹⁸, and Ser⁵²² (14, 15).CRMPs are also substrates of several tyrosine kinases. The phosphorylation of CRMP2 by Fes/Fps and Fer has been shown to be involved in Sema3A signaling (16, 17). Phosphorylation of CRMP2 at Tyr⁴⁷⁹ by a Src family tyrosine kinase Yes regulates CXCL12-induced T lymphocyte migration (18). We reported previously that Fyn is involved in Sema3A signaling (19). Fyn associates with PlexinA2, one of the components of the Sema3A receptor complex. Fyn also activates Cdk5 through the phosphorylation at Tyr¹⁵ of Cdk5 (19). In dorsal root ganglion (DRG) neurons from fyn-deficient mice, Sema3A-induced growth cone collapse response is attenuated compared with control mice (19). Furthermore, we recently found that Fyn phosphorylates CRMP1 and that this phosphorylation is involved in Reelin signaling (4). Although it has been shown that CRMP2 is involved in Sema3A signaling (1, 11, 13), the relationship between Fyn and CRMP2 in Sema3A signaling and the tyrosine phosphorylation site(s) of CRMPs remain unknown.Here, we show that Fyn phosphorylates CRMP2 at Tyr³². Using a phospho-specific antibody against Tyr³², we determined that the residue is phosphorylated in vivo. A nonphosphorylated mutant CRMP2Y32F inhibits Sema3A-induced growth cone collapse. These results indicate that tyrosine phosphorylation by Fyn at Tyr³² is involved in Sema3A signaling.     

In-depth Qualitative and Quantitative Profiling of Tyrosine Phosphorylation Using a Combination of Phosphopeptide Immunoaffinity Purification and Stable Isotope Dimethyl Labeling

Several mass spectrometry-based assays have emerged for the quantitative profiling of cellular tyrosine phosphorylation. Ideally, these methods should reveal the exact sites of tyrosine phosphorylation, be quantitative, and not be cost-prohibitive. The latter is often an issue as typically several milligrams of (stable isotope-labeled) starting protein material are required to enable the detection of low abundance phosphotyrosine peptides. Here, we adopted and refined a peptidecentric immunoaffinity purification approach for the quantitative analysis of tyrosine phosphorylation by combining it with a cost-effective stable isotope dimethyl labeling method. We were able to identify by mass spectrometry, using just two LC-MS/MS runs, more than 1100 unique non-redundant phosphopeptides in HeLa cells from about 4 mg of starting material without requiring any further affinity enrichment as close to 80% of the identified peptides were tyrosine phosphorylated peptides. Stable isotope dimethyl labeling could be incorporated prior to the immunoaffinity purification, even for the large quantities (mg) of peptide material used, enabling the quantification of differences in tyrosine phosphorylation upon pervanadate treatment or epidermal growth factor stimulation. Analysis of the epidermal growth factor-stimulated HeLa cells, a frequently used model system for tyrosine phosphorylation, resulted in the quantification of 73 regulated unique phosphotyrosine peptides. The quantitative data were found to be exceptionally consistent with the literature, evidencing that such a targeted quantitative phosphoproteomics approach can provide reproducible results. In general, the combination of immunoaffinity purification of tyrosine phosphorylated peptides with large scale stable isotope dimethyl labeling provides a cost-effective approach that can alleviate variation in sample preparation and analysis as samples can be combined early on. Using this approach, a rather complete qualitative and quantitative picture of tyrosine phosphorylation signaling events can be generated.Reversible tyrosine phosphorylation plays an important role in numerous cellular processes like growth, differentiation, and migration. Phosphotyrosine signaling is tightly controlled by the balanced action of protein-tyrosine kinases and protein-tyrosine phosphatases. Aberrant tyrosine phosphorylation has been suggested to be an underlying cause in multiple cancers (1). Therefore, the identification of tyrosine phosphorylated proteins and the investigation into their involvement in signaling pathways are important. Several groups have attempted to comprehensively study tyrosine phosphorylation by proteomics means (2–5). However, large scale identification of tyrosine phosphorylation sites by MS can be hindered by the low abundance of tyrosine phosphorylated proteins. Especially, signaling intermediates are usually low abundance proteins that show substoichiometric phosphorylation levels. In addition, the identification by mass spectrometry of phosphopeptides from a complex cellular lysate digest is often complicated by ion suppression effects due to a high background of non-phosphorylated peptides. Enrichment of tyrosine phosphorylated proteins or peptides prior to mass spectrometric detection is therefore essential. Traditionally, antibodies against phosphorylated tyrosine have been used to immunoprecipitate tyrosine phosphorylated proteins from cultured cells (2–4, 6–8). This phosphoprotein immunoaffinity purification method has for example been used to study the global dynamics of phosphotyrosine signaling events after EGF¹ stimulation using stable isotope labeling by amino acids in cell culture (SILAC) (2). This approach led to the identification of known and previously unidentified signaling proteins in the EGF receptor (EGFR) pathway, including their temporal activation profile after stimulation of the EGFR, providing crucial information for modeling signaling events in the cell. However, as the identification and quantification of these phosphorylated proteins in these studies were not necessarily based on tyrosine phosphorylated peptides but largely on non-phosphorylated peptides, little information is derived on the exact site(s) of tyrosine phosphorylation. Also, binding partners of tyrosine phosphorylated proteins, which themselves are not tyrosine phosphorylated, might be co-precipitated and impair the tyrosine phosphorylation quantification. Immunoaffinity purification of phosphotyrosine peptides, rather than proteins, using anti-phosphotyrosine antibodies (5, 9–16) significantly facilitates the identification of the site(s) of phosphorylation as it greatly alleviates most of the above mentioned problems because the tyrosine phosphorylated site can be directly identified and quantified.Accurate MS-based quantification is typically performed by stable isotope labeling. The isotopes can be incorporated metabolically during cell culture as in SILAC (17) or chemically as in an isobaric tag for relative and absolute quantitation (iTRAQ) (18) or stable isotope dimethyl labeling (19–21). Typically, the most precise quantification can be obtained by metabolic labeling as the different samples can be combined at the level of intact cells (22). However, metabolic labeling is somewhat limited to biological systems that can be grown in culture, and the medium may have an effect on the growth and development of the cells. iTRAQ has been used in conjunction with phosphotyrosine peptide immunoprecipitation (5). As the chemical labeling is performed before immunoprecipitation, the differentially labeled samples can be precipitated together, thereby neutralizing the potentially largest source of variation. However, as this phosphotyrosine peptide immunoprecipitation is typically performed on several hundreds of micrograms to milligrams of protein sample, iTRAQ provides in these cases a rather cost-prohibitive means.Here, we present an optimized immunoaffinity purification approach for the analysis of tyrosine phosphorylation combined with stable isotope dimethyl labeling (19–21, 23). We efficiently enriched and identified by MS 1112 unique phosphopeptides derived from 4 mg of starting protein material without any further affinity chromatographic enrichment whereby up to 80% of the peptides analyzed in the final LC run were phosphotyrosine peptides. We further advanced the method by introducing triplex stable isotope dimethyl labeling prior to immunoprecipitation. We quantified differences in tyrosine phosphorylation upon pervanadate treatment or EGF stimulation to detect site-specific changes in tyrosine phosphorylation. 128 unique phosphotyrosine peptides were identified and quantified upon pervanadate treatment. By using an internal standard comprising both mock and pervanadate-treated samples, we could more confidently identify and quantify phosphorylation sites that are strongly regulated and on-off situations. Analysis of EGF-stimulated HeLa cells resulted in the quantification of 73 unique phosphotyrosine peptides. Most of the up-regulated phosphotyrosine peptides that were identified have been reported previously to be involved in the EGFR signaling pathway, validating our approach. However, for the first time, we found TFG to also become highly tyrosine phosphorylated upon EGF stimulation together with some tyrosine phosphorylation sites on for example IRS2, SgK269, and DLG3 that have not been firmly established earlier to be involved in EGFR signaling.In general, we show that the combination of immunoaffinity purification of tyrosine phosphorylated peptides with large scale chemical stable isotope dimethyl labeling provides a cost-effective approach that can alleviate variation in immunoprecipitation and LC-MS as samples can be combined before immunoprecipitation and the necessity of performing additional enrichment is removed by an optimization of the protocol. With only a single LC-MS run, already a rather complete qualitative and quantitative picture of a signaling event can be generated.     

Phosphoproteomics of Klebsiella pneumoniae NTUH-K2044 Reveals a Tight Link between Tyrosine Phosphorylation and Virulence

Encapsulated Klebsiella pneumoniae is the predominant causative agent of pyogenic liver abscess, an emerging infectious disease that often complicates metastatic meningitis or endophthalmitis. The capsular polysaccharide on K. pneumoniae surface was determined as the key to virulence. Although the regulation of capsular polysaccharide biosynthesis is largely unclear, it was found that protein-tyrosine kinases and phosphatases are involved. Therefore, the identification and characterization of such kinases, phosphatases, and their substrates would advance our knowledge of the underlying mechanism in capsule formation and could contribute to the development of new therapeutic strategies. Here, we analyzed the phosphoproteome of K. pneumoniae NTUH-K2044 with a shotgun approach and identified 117 unique phosphopeptides along with 93 in vivo phosphorylated sites corresponding to 81 proteins. Interestingly, three of the identified tyrosine phosphorylated proteins, namely protein-tyrosine kinase (Wzc), phosphomannomutase (ManB), and undecaprenyl-phosphate glycosyltransferase (WcaJ), were found to be distributed in the cps locus and thus were speculated to be involved in the converging signal transduction of capsule biosynthesis. Consequently, we decided to focus on the lesser studied ManB and WcaJ for mutation analysis. The capsular polysaccharides of WcaJ mutant (WcaJY5F) were dramatically reduced quantitatively, and the LD₅₀ increased by 200-fold in a mouse peritonitis model compared with the wild-type strain. However, the capsular polysaccharides of ManB mutant (ManBY26F) showed no difference in quantity, and the LD₅₀ increased by merely 6-fold in mice test. Our study provided a clear trend that WcaJ tyrosine phosphorylation can regulate the biosynthesis of capsular polysaccharides and result in the pathogenicity of K. pneumoniae NTUH-K2044.Protein phosphorylation is one of the most biologically relevant and ubiquitous post-translational modifications in both eukaryotic and prokaryotic organisms. It is best known that protein phosphorylation is a reversible enzyme-catalyzed process that is controlled by various kinases and phosphatases. The aberrant functions often result in irregular protein phosphorylation and ultimately lead to serious disease states such as malignant transformation, immune disorders, and pathogenic infections in mammals (1, 2). Recently, accumulating evidences suggest that Ser/Thr/Tyr phosphorylations also contribute to regulate a diverse range of cellular responses and physiological processes in prokaryotes (1). Among them, tyrosine phosphorylation in encapsulated bacteria has been discovered to play key roles in capsular polysaccharide (CPS1; K antigen) biosynthesis, which leads to virulence (3, 4). This thick layer of exopolysaccharide on many pathogenic bacteria can act as a physical boundary to evade phagocytosis and complement-mediated killing and further inhibit complement activation of the host (1, 5, 6).In 1996, Acinetobacter johnsonii protein-tyrosine kinase (Ptk) was first discovered and categorized under the bacterial protein-tyrosine kinase (BY-kinase) family (1, 7, 8). Shortly after, its function in bacterial exopolysaccharide production and transport was characterized (1, 7, 8). From then on, many more bacterial tyrosine kinases such as Wzc of Escherichia coli (1, 9) and EpsB of Pseudomonas solanacearum (10, 11) were found to possess this conserved property; deletion of such tyrosine kinases will result in the loss of exopolysaccharide production (12). Therefore, several experiments were conducted to investigate the role of the downstream substrates of the tyrosine kinases in different strains of bacteria, and some targeted proteins were found to participate in the exopolysaccharide anabolism (13, 14). These findings demonstrated a direct relationship between bacterial tyrosine phosphorylation and exopolysaccharide biosynthesis that was directly reflected in the strain virulence.In the past, the functional roles of the critical components involved in protein phosphorylation were defined by basic biochemical and genetic approaches (1). However, there exists a salient gap between the growing number of identified protein-tyrosine kinases/phosphatases and the relative paucity of protein substrates characterized to date. Genomic sequence analyses and advanced high resolution/high accuracy MS systems with vastly improved phosphopeptide enrichment strategies are among the two key enabling technologies that allow a high efficiency identification of the scarcely detectable site-specific phosphorylations in bacterial systems (15). Mann et al. (16) were the first to initiate a systematic study of the phosphoproteome of B. subtilis in 2007 followed by similar site-specific phosphoproteomics analyses of E. coli (17), Lactococcus lactis (18), and Halobacterium salinarum (19). These pioneering works have since set the foundation in bacterial phosphoproteomics but have not been specifically carried out to address a particular biological issue of causal relevance to virulence or pathogenesis.Klebsiella pneumoniae is a Gram-negative, non-motile, facultative anaerobic, and rod-shaped bacterium. It is commonly found in water and soil (20) as well as on plants (21) and mucosal surfaces of mammals, such as human, horse, and swine (22, 23). It was demonstrated that CPS on the surface of K. pneumoniae is the prime factor of virulence and toxicity in causing pyogenic liver abscess (PLA), a common intra-abdominal infection with a high 10–30% mortality rate worldwide (24–29). There are also variations in virulence in regard to different capsular serotypes; K1 and K2 were found to be especially pathogenic in causing PLA in a mouse model (30) compared with other serotypes, which show little or no effect (31–34). The K. pneumoniae NTUH-K2044 (K2044) strain, encapsulated with K1 antigen (35), was isolated from clinical K. pneumoniae liver abscess patients. It has become an important emerging pathogen (36) because it usually complicates metastatic septic endophthalmitis and irreversible central nervous system infections independent of host underlying diseases (30, 34). The transmission rate is high (37), and it often rapidly leads to outbreaks of community-acquired infections, such as bacteremia, nosocomial pneumonia, and sepsis, common in immunocompromised individuals (38).In this study, we wanted to prove that the biosynthesis of CPS is mediated through tyrosine phosphorylation of a subset of proteins. An MS-based systematic phosphoproteomics analysis was conducted on K2044 to identify tyrosine phosphorylated proteins that are also associated with CPS biosynthesis. We further validated the relationship between tyrosine phosphorylation on those proteins and virulence of K2044 by site-directed mutagenesis, CPS quantification, serum killing, and mouse lethality assay.     

Tyrosine Phosphorylation of Growth Factor Receptor-bound Protein-7 by Focal Adhesion Kinase in the Regulation of Cell Migration, Proliferation, and Tumorigenesis

We have previously reported that growth factor receptor-bound protein-7 (Grb7), an Src-homology 2 (SH2)-containing adaptor protein, enables interaction with focal adhesion kinase (FAK) to regulate cell migration in response to integrin activation. To further elucidate the signaling events mediated by FAK·Grb7 complexes in promoting cell migration and other cellular functions, we firstly examined the phos pho ryl a ted tyrosine site(s) of Grb7 by FAK using an in vivo mutagenesis. We found that FAK was capable of phos pho rylating at least 2 of 12 tyrosine residues within Grb7, Tyr-188 and Tyr-338. Moreover, mutations converting the identified Tyr to Phe inhibited integrin-dependent cell migration as well as impaired cell proliferation but not survival compared with the wild-type control. Interestingly, the above inhibitory effects caused by the tyrosine phos pho ryl a tion-deficient mutants are probably attributed to their down-regulation of phospho-Tyr-397 of FAK, thereby implying a mechanism by competing with wild-type Grb7 for binding to FAK. Consequently, these tyrosine phos pho ryl a tion-deficient mutants evidently altered the phospho-Tyr-118 of paxillin and phos pho ryl a tion of ERK1/2 but less on phospho-Ser-473 of AKT, implying their involvement in the FAK·Grb7-mediated cellular functions. Additionally, we also illustrated that the formation of FAK·Grb7 complexes and Grb7 phos pho ryl a tion by FAK in an integrin-dependent manner were essential for cell migration, proliferation and anchorage-independent growth in A431 epidermal carcinoma cells, indicating the importance of FAK·Grb7 complexes in tumorigenesis. Our data provide a better understanding on the signal transduction event for FAK·Grb7-mediated cellular functions as well as to shed light on a potential therapeutic in cancers.Growth factor receptor bound protein-7 (Grb7)² is initially identified as a SH2 domain-containing adaptor protein bound to the activated EGF receptor (1). Grb7 is composed of an N-terminal proline-rich region, following a putative RA (Ras-associating) domain and a central PH (pleckstrin homology) domain and a BPS motif (between PH and SH2 domains), and a C-terminal SH2 domain (2–6). Despite the lack of enzymatic activity, the presence of multiple protein-protein interaction domains allows Grb7 family adaptor proteins to participate in versatile signal transduction pathways and, therefore, to regulate many cellular functions (4–6). A number of signaling molecules has been reported to interact with these featured domains, although most of the identified Grb7 binding partners are mediated through its SH2 domain. For example, the SH2 domain of Grb7 has been demonstrated to be capable of binding to the phospho-tyrosine sites of EGF receptor (1), ErbB2 (7), ErbB3 and ErbB4 (8), Ret (9), platelet-derived growth factor receptor (10), insulin receptor (11), SHPTP2 (12), Tek/Tie2 (13), caveolin (14), c-Kit (15), EphB1 (16), G6f immunoreceptor protein (17), Rnd1 (18), Shc (7), FAK (19), and so on. The proceeding α-helix of the PH domain of Grb7 is the calmodulin-binding domain responsible for recruiting Grb7 to plasma membrane in a Ca²⁺-dependent manner (20), and the association between the PH domain of Grb7 and phosphoinositides is required for the phosphorylation by FAK (21). Two additional proteins, NIK (nuclear factor κB-inducing kinase) and FHL2 (four and half lim domains isoform 2), in association with the GM region (Grb and Mig homology region) of Grb7 are also reported, although the physiological functions for these interactions remain unknown (22, 23). Recently, other novel roles in translational controls and stress responses through the N terminus of Grb7 are implicated for the findings of Grb7 interacting with the 5′-untranslated region of capped targeted KOR (kappa opioid receptor) mRNA and the Hu antigen R of stress granules in an FAK-mediated phosphorylation manner (24, 25).Unlike its member proteins Grb10 and Grb14, the role of Grb7 in cell migration is unambiguous and well documented. This is supported by a series of studies. Firstly, Grb7 family members share a significantly conserved molecular architecture with the Caenorhabditis elegans Mig-10 protein, which is involved in neuronal cell migration during embryonic development (4, 5, 26), suggesting that Grb7 may play a role in cell migration. Moreover, Grb7 is often co-amplified with Her2/ErbB2 in certain human cancers and tumor cell lines (7, 27, 28), and its overexpression resulted in invasive and metastatic consequences of various cancers and tumor cells (23, 29–33). On the contrary, knocking down Grb7 by RNA interference conferred to an inhibitory outcome of the breast cancer motility (34). Furthermore, interaction of Grb7 with autophosphorylated FAK at Tyr-397 could promote integrin-mediated cell migration in NIH 3T3 and CHO cells, whereas overexpression of its SH2 domain, an dominant negative mutant of Grb7, inhibited cell migration (19, 35). Recruitment and phosphorylation of Grb7 by EphB1 receptors enhanced cell migration in an ephrin-dependent manner (16). Recently, G7–18NATE, a selective Grb7-SH2 domain affinity cyclic peptide, was demonstrated to efficiently block cell migration of tumor cells (32, 36). In addition to cell migration, Grb7 has been shown to play a role in a variety of physiological and pathological events, for instance, kidney development (37), tumorigenesis (7, 14, 38–41), angiogenic activity (20), proliferation (34, 42, 43), anti-apoptosis (44), gene expression regulation (24), Silver-Russell syndrome (45), rheumatoid arthritis (46), atopic dermatitis (47), and T-cell activation (17, 48). Nevertheless, it remains largely unknown regarding the downstream signaling events of Grb7-mediated various functions. In particular, given the role of Grb7 as an adaptor molecule and its SH2 domain mainly interacting with upstream regulators, it will be interesting to identify potential downstream effectors through interacting with the functional GM region or N-terminal proline-rich region.In this report, we identified two tyrosine phosphorylated sites of Grb7 by FAK and deciphered the signaling targets downstream through these phosphorylated tyrosine sites to regulate various cellular functions such as cell migration, proliferation, and survival. In addition, our study sheds light on tyrosine phosphorylation of Grb7 by FAK involved in tumorigenesis.     

Identification of Extracellular Signal-regulated Kinase 1 (ERK1) Direct Substrates using Stable Isotope Labeled Kinase Assay-Linked Phosphoproteomics

Kinase mediated phosphorylation signaling is extensively involved in cellular functions and human diseases, and unraveling phosphorylation networks requires the identification of substrates targeted by kinases, which has remained challenging. We report here a novel proteomic strategy to identify the specificity and direct substrates of kinases by coupling phosphoproteomics with a sensitive stable isotope labeled kinase reaction. A whole cell extract was moderately dephosphorylated and subjected to in vitro kinase reaction under the condition in which ¹⁸O-ATP is the phosphate donor. The phosphorylated proteins are then isolated and identified by mass spectrometry, in which the heavy phosphate (+85.979 Da) labeled phosphopeptides reveal the kinase specificity. The in vitro phosphorylated proteins with heavy phosphates are further overlapped with in vivo kinase-dependent phosphoproteins for the identification of direct substrates with high confidence. The strategy allowed us to identify 46 phosphorylation sites on 38 direct substrates of extracellular signal-regulated kinase 1, including multiple known substrates and novel substrates, highlighting the ability of this high throughput method for direct kinase substrate screening.Protein phosphorylation regulates almost all aspects of cell life, such as cell cycle, migration, and apoptosis (1), and deregulation of protein phosphorylation is one of the most frequent causes or consequences of human diseases including cancers, diabetes, and immune disorders (2). Up till now, however, known substrates are far from saturation for the majority of protein kinases (3); thus, mapping comprehensive kinase-substrate relationships is essential to understanding biological mechanisms and uncovering new drug targets (4).Accompanied with advances of high-speed and high-resolution mass spectrometry, the technique of kinase substrate screening using proteomic strategy is quickly evolving (5–7). Mass spectrometry has been extensively used for kinase-substrate interaction mapping (8) and global phosphorylation profiling (9). Although thousands of phosphorylation sites have been detected, complex phosphorylation cascade and crosstalk between pathways make it difficult for large-scale phosphoproteomics to reveal direct relationships between protein kinases and their substrates (10, 11). Extensive statistics, bioinformatics, and downstream biochemical assays are mandatory for the substrate verification (12, 13). Another strategy uses purified, active kinases to phosphorylate cell extracts in vitro, followed by mass spectrometric analysis to identify phosphoproteins. This approach inevitably faces the major challenge of separating real sites phosphorylated by target kinase and the phosphorylation triggered by endogenous kinases from cell lysates (14). Analog-sensitive kinase allele (15) overcomes the issue by utilizing the engineered kinase that can exclusively take a bulky-ATP analog under the reaction condition. Analog-sensitive kinase allele has been coupled with γ-thiophosphate analog ATP to facilitate the mass spectrometric analysis (16–18).We have introduced kinase assay-linked phosphoproteomics (KALIP)¹ to link the in vitro substrate identification and physiological phosphorylation events together in a high throughput manner (19, 20). The strategy, however, has only been applied to identify direct substrates of tyrosine kinases. In this study, we expanded the application of KALIP to serine/threonine kinases by introducing a quantitative strategy termed Stable Isotope Labeled Kinase Assay-Linked Phosphoproteomics (siKALIP). The method was applied to identify direct substrates of extracellular signal-regulated kinase 1 (ERK1), a serine/threonine kinase acting as an essential component of the Mitogen-activated protein kinase (MAPK) signal transduction pathway (21). A defect in the MAP/ERK pathway causes uncontrolled growth, which likely leads to cancer (22) and other diseases (23–25). ERK1 can be activated by growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and nerve growth factor (NGF) (26). Upon stimulation, ERK1 phosphorylates hundreds of substrates in various cellular compartments including cytoplasm, nucleus, and membrane (27). Among 38 ERK1 direct substrates identified by siKALIP, more than one third are previously discovered by classical molecular biology approaches, highlighting high specificity and sensitivity of the strategy. The results also support the hypothesis that ERK1 plays complex roles in multiple pathways that are essential for the cell growth regulation.     

Evolutionary Constraints of Phosphorylation in Eukaryotes,Prokaryotes, and Mitochondria

High accuracy mass spectrometry has proven to be a powerful technology for the large scale identification of serine/threonine/tyrosine phosphorylation in the living cell. However, despite many described phosphoproteomes, there has been no comparative study of the extent of phosphorylation and its evolutionary conservation in all domains of life. Here we analyze the results of phosphoproteomics studies performed with the same technology in a diverse set of organisms. For the most ancient organisms, the prokaryotes, only a few hundred proteins have been found to be phosphorylated. Applying the same technology to eukaryotic species resulted in the detection of thousands of phosphorylation events. Evolutionary analysis shows that prokaryotic phosphoproteins are preferentially conserved in all living organisms, whereas-site specific phosphorylation is not. Eukaryotic phosphosites are generally more conserved than their non-phosphorylated counterparts (with similar structural constraints) throughout the eukaryotic domain. Yeast and Caenorhabditis elegans are two exceptions, indicating that the majority of phosphorylation events evolved after the divergence of higher eukaryotes from yeast and reflecting the unusually large number of nematode-specific kinases. Mitochondria present an interesting intermediate link between the prokaryotic and eukaryotic domains. Applying the same technology to this organelle yielded 174 phosphorylation sites mapped to 74 proteins. Thus, the mitochondrial phosphoproteome is similarly sparse as the prokaryotic phosphoproteomes. As expected from the endosymbiotic theory, phosphorylated as well as non-phosphorylated mitochondrial proteins are significantly conserved in prokaryotes. However, mitochondrial phosphorylation sites are not conserved throughout prokaryotes, consistent with the notion that serine/threonine phosphorylation in prokaryotes occurred relatively recently in evolution. Thus, the phosphoproteome reflects major events in the evolution of life.Reversible protein phosphorylation on serines, threonines, and tyrosines plays a crucial role in regulating processes in all living organisms ranging from prokaryotes to eukaryotes (1). Traditionally, phosphorylation has been detected in single, purified proteins using in vitro assays. Recent advances in mass spectrometry (MS)-based proteomics now allow the identification of in vivo phosphorylation sites with high accuracy (2–7). On-line databases such as PhosphoSite (8), Phospho.ELM (9), and PHOSIDA1 (10) have collected and organized thousands of identified phosphosites. These databases as well as dedicated analysis environments such as NetworKIN (11, 12) offer and use contextual information including structural features, potential kinases, and conservation. They constitute resources that should allow the derivation of general patterns for phosphorylation events. Specifically, the recent availability of data for archaeal, prokaryotic, and diverse eukaryotic phosphoproteomes in these databases should enable investigation of the evolutionary history of this post-translational modification.Prokaryotes have two separate classes of phosphorylation events. Apart from the canonical histidine/aspartate phosphorylation, which has been studied for decades, serine/threonine/tyrosine phosphorylation is also present and has recently become amenable to analysis by MS (13). Bacterial phosphoproteins are involved in protein synthesis, carbohydrate metabolism, and the phosphoenolpyruvate-dependent phosphotransferase system. Recent phosphoproteomics studies of Bacillus subtilis, Escherichia coli, and Lactococcus lactis described around 100 phosphorylation sites on serine, threonine, and tyrosine in each of these species (13–15). Bacterial phosphorylation sites can change in response to environmental conditions (16).Interestingly, even archaea have serine/threonine and tyrosine phosphorylation. A recent study of Halobacterium salinarum described 75 serine/threonine/tyrosine phosphorylation sites on 62 proteins involved in a wide range of cellular processes including a variety of metabolic pathways (17).Although only a few hundred phosphorylation events have been found in prokaryotic species, similar experimental conditions and effort have yielded the detection of thousands of phosphorylation events in eukaryotes ranging from yeast to human (7, 18–21). Before the advent of large scale phosphoproteomics, serine/threonine/tyrosine phosphorylation has been estimated to affect one-third of all proteins (22). Recent large scale phosphoproteomics studies now suggest that more than half of all eukaryotic proteins are phosphorylated (23).A key event in evolution was the endosymbiosis of prokaryotes that enabled the development of a much more complex type of life, the eukaryotic cell. Analyses of mitochondrial genes suggest that the α-proteobacterium Rickettsia prowazekii is the endosymbiotic precursor leading to modern mitochondria (24). Almost all of the mitochondrial genes have migrated to the nuclear genome during subsequent evolution, and it is predicted that 10–15% of eukaryotic nuclear genes of organisms encode mitochondrial proteins (25).Thus, mitochondria with their unique evolutionary position between prokaryotes and eukaryotes form an interesting link for the evolutionary analysis of phosphorylation. Several studies investigated the mitochondrial phosphoproteome in different organisms using gel electrophoresis or specific enrichment methods coupled with mass spectrometry (26–28). Those studies established potential mitochondrial phosphoproteins. Three large scale studies based on affinity enrichment of phosphopeptides and mass spectrometry obtained direct experimental evidence of phosphorylation sites in mitochondria. Lee et al. (29) used a combination of different peptide enrichment strategies and found 80 phosphorylation sites of 48 different proteins from mouse liver. Very recently, a study by Deng et al. (30) characterized the murine cardiac mitochondrial mouse phosphoproteome, covering 236 phosphosites on 181 proteins. Investigating yeast, Reinders et al. (31) assigned 84 phosphorylation sites in 62 proteins.To enable comparative analysis of phosphoproteomes between all domains of life and mitochondria, here we experimentally determined a high accuracy mitochondrial mouse phosphoproteome based on technology conditions similar to those applied to the identification of prokaryotic and eukaryotic phosphoproteomes. We then performed a detailed evolutionary study of the conservation of the identified phosphoproteins and phosphorylation sites in prokaryotes and in eukaryotes. This allowed an initial comparison of the phosphoproteomes of prokaryotes, mitochondria, and eukaryotes.     

B Cell Receptor-induced Phosphorylation of Pyk2 and Focal Adhesion Kinase Involves Integrins and the Rap GTPases and Is Required for B Cell Spreading

Signaling by the B cell receptor (BCR) promotes integrin-mediated adhesion and cytoskeletal reorganization. This results in B cell spreading, which enhances the ability of B cells to bind antigens and become activated. Proline-rich tyrosine kinase (Pyk2) and focal adhesion kinase (FAK) are related cytoplasmic tyrosine kinases that regulate cell adhesion, cell morphology, and cell migration. In this report we show that BCR signaling and integrin signaling collaborate to induce the phosphorylation of Pyk2 and FAK on key tyrosine residues, a modification that increases the kinase activity of Pyk2 and FAK. Activation of the Rap GTPases is critical for BCR-induced integrin activation as well as for BCR- and integrin-induced reorganization of the actin cytoskeleton. We now show that Rap activation is essential for BCR-induced phosphorylation of Pyk2 and for integrin-induced phosphorylation of Pyk2 and FAK. Moreover Rap-dependent phosphorylation of Pyk2 and FAK required an intact actin cytoskeleton as well as actin dynamics, suggesting that Rap regulates Pyk2 and FAK via its effects on the actin cytoskeleton. Importantly B cell spreading induced by BCR/integrin co-stimulation or by integrin engagement was inhibited by short hairpin RNA-mediated knockdown of either Pyk2 or FAK expression and by treatment with PF-431396, a chemical inhibitor that blocks the kinase activities of both Pyk2 and FAK. Thus Pyk2 and FAK are downstream targets of the Rap GTPases that play a key role in regulating B cell morphology.Antibodies (Abs)² made by B lymphocytes play a critical role in host defense against infection. Antigen-induced signaling by the B cell receptor (BCR) initiates an activation program that leads to B cell proliferation and subsequent differentiation into Ab-producing cells. BCR clustering by antigens or by anti-immunoglobulin (anti-Ig) Abs used as surrogate antigens initiates multiple signaling pathways that control gene expression, cell survival, and proliferation pathways (1–3).BCR signaling also promotes integrin activation (4, 5), localized actin polymerization, reorganization of the actin cytoskeleton, and changes in B cell morphology (6, 7), all of which may facilitate B cell activation. Integrin activation and cell spreading is critical for the activation of B cells by membrane-bound antigens. Macrophages, dendritic cells, and follicular dendritic cells can present arrays of captured antigens to B cells (8, 9), and this may be one of the main ways in which B cells encounter antigens (10). BCR-induced integrin activation prolongs the interaction between the B cell and the antigen-presenting cell and also allows the B cell to spread on the surface of the antigen-presenting cell such that more BCRs can encounter and bind membrane-bound antigens (11). Subsequent contraction of the B cell membrane allows the B cells to gather the BCR-bound antigen into an immune synapse in which clustered antigen-engaged BCRs are surrounded by a ring of ligand-bound integrins. Formation of this immune synapse reduces the amount of antigen that is required for B cell activation (12, 13).Recent work has shown that B cells in lymphoid organs may contact soluble antigens by extending membrane processes into a highly organized network of lymph-filled conduits (14). These conduits are created by fibroblastic reticular cells that partially ensheathe collagen fibrils. In addition to being rich in collagen, fibronectin, and other extracellular matrix (ECM) components, the fibroblastic reticular cells that form these conduits express high levels of intercellular adhesion molecule-1, the ligand for the α_Lβ₂ integrin (lymphocyte function-associated antigen-1 (LFA-1)) on B cells (10). Thus B cells interacting with these conduits are likely to be in contact with integrin ligands, and integrin-dependent spreading may enhance the ability of B cells to extend membrane processes into the fibroblastic reticular cell conduit.In addition to promoting cell spreading, integrins can act as co-stimulatory receptors that enhance signaling by many receptors including the T cell receptor and the BCR (15–17). Thus signaling proteins that regulate B cell spreading and that are also targets of BCR/integrin co-stimulation may play a key role in the activation of B cells by membrane-bound antigens as well as soluble antigens that are delivered to lymphoid organs by fibroblastic reticular cell conduits.Proline-rich tyrosine kinase (Pyk2) and focal adhesion kinase (FAK) are related non-receptor protein-tyrosine kinases that integrate signals from multiple receptors and play an important role in regulating cell adhesion, cell morphology, and cell migration in many cell types (18–20). Integrins, receptor tyrosine kinases, antigen receptors, and G protein-coupled chemokine receptors all stimulate tyrosine phosphorylation of Pyk2 and FAK, a modification that increases the enzymatic activity of these kinases and allows them to bind SH2 domain-containing signaling proteins (21). FAK, which is expressed in almost all tissues (21), is a focal adhesion component that mediates integrin-dependent cell migration (22), cell spreading, and cell adhesion (18) in adherent cells as well as co-clustering of LFA-1 with the T cell receptor in lymphocytes (23). Pyk2 is expressed mainly in hematopoietic cells, osteoclasts, and the central nervous system (24) and is critical for chemokine-induced migration of B cells, macrophages, and natural killer cells (20, 25, 26) as well as the spreading of osteoclasts on vitronectin (27). FAK and Pyk2 are thought to mediate overlapping but distinct functions because Pyk2 expression only partially reverses the cell adhesion and migration defects in FAK-deficient fibroblasts (28).In B cells, clustering of the BCR, β₁ integrins, or β₇ integrins induces tyrosine phosphorylation of both Pyk2 and FAK (29–33). FAK is involved in the chemokine-induced adhesion of B cell progenitors (34), and Pyk2 is required for chemokine-induced migration of mature B cells (25). However, the role of these kinases in BCR- and integrin-induced B cell spreading has not been investigated, and the signaling pathways that link the BCR and integrins to tyrosine phosphorylation of Pyk2 and FAK have not been elucidated.We have shown previously that the ability of the BCR to induce integrin activation, B cell spreading, and immune synapse formation requires activation of the Rap GTPases (6, 17). In addition to binding effector proteins such as RapL and Rap1-interacting adaptor molecule (RIAM) that promote integrin activation (35–37), the active GTP-bound forms of Rap1 and Rap2 bind multiple proteins that control actin dynamics and cell morphology (38). Moreover we showed that BCR/integrin-induced phosphorylation of Pyk2 in B cells is dependent on Rap activation (17). However, this previous study did not address how Rap-GTP links the BCR and integrins to Pyk2 phosphorylation, whether Rap activation is important for FAK phosphorylation in B cells, or whether B cell spreading is regulated by Pyk2 or FAK. We now show that Pyk2 and FAK are differentially expressed and localized in B cells, that Pyk2 and FAK are important for B cell spreading, and that integrin engagement enhances BCR-induced phosphorylation of Pyk2 and FAK, a process that depends on both Rap activation and actin dynamics.     

Rescue of a Trafficking Defective Human Pacemaker Channel via a Novel Mechanism: ROLES OF Src, Fyn, AND Yes TYROSINE KINASES*

Therapeutic strategies such as using channel blockers and reducing culture temperature have been used to rescue some long QT-associated voltage-gated potassium Kv trafficking defective mutant channels. A hyperpolarization-activated cyclic nucleotide-gated HCN4 pacemaker channel mutant (D553N) has been recently found in a patient associated with cardiac arrhythmias including long QT. D553N showed the defective trafficking to the cell surface, leading to little ionic current expression (loss-of-function). We show in this report that enhanced tyrosine phosphorylation mediated by Src, Fyn, and Yes kinases was able to restore the surface expression of D553N for normal current expression. Src or Yes, but not Fyn, significantly increased the current density and surface expression of D553N. Fyn accelerated the activation kinetics of the rescued D553N. Co-expression of D553N with Yes exhibited the slowest activation kinetics of D553N. Src, Fyn, and Yes significantly enhanced the tyrosine phosphorylation of D553N. A combination of Src, Fyn, and Yes rescued the current expression and the gating of D553N comparable with those of wild-type HCN4. In conclusion, we demonstrate a novel mechanism using three endogenous Src kinases to rescue a trafficking defective HCN4 mutant channel (D553N) by enhancing the tyrosine phosphorylation of the mutant channel protein.Defective trafficking leading to the reduced surface expression of ion channels is one of the mechanisms responsible for a loss-of-function of the ion channel on the plasma membrane (1). Several methods have been developed to rescue the voltage-gated potassium Kv trafficking defective channels: reducing the culture temperature, applying the channel blockers, altering the molar ratio of glycerol, and using the sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin (2–6).Hyperpolarizing-activated cyclic nucleotide-gated (HCN)³ pacemaker channels generate time- and voltage-dependent inward currents, named I_h in neurons or I_f in the heart (7). They are important in various cell functions including excitability, synapse transmission, and rhythmic activity (7). The most well studied regulation of I_f is its response to autonomic stimulation. β-Adrenergic receptor activation increases and acetylcholine receptor activation decreases the intracellular cAMP levels, which in turn increases/decreases I_f by binding to the cyclic nucleotide-binding domain of the HCN channels, respectively (7). Other important mechanisms for the modulation of I_f/HCN channels have recently been found including β-subunit (8), lipids (9, 10), and p38 mitogen-activated protein kinase (11).Accumulating evidence has revealed tyrosine phosphorylation as an important mechanism for modulation of HCN channel properties (12–16). An acute increase in tyrosine phosphorylation of I_f or HCN channels increases the channel activity, including an increase in the current amplitude, a positive shift of the voltage-dependent activation, an acceleration of activation kinetics, and an increase in whole cell conductance (12–15). Recently, we discovered that the cell surface expression of HCN2 channels can be remarkably inhibited by tyrosine dephosphorylation mediated by receptor-like protein tyrosine phosphatase α (RPTPα) and increased by tyrosine phosphorylation via Src kinase after long term treatment (17).D553N, a missense HCN4 mutant, was recently identified in a patient with cardiac arrhythmia associated with depressed HCN gating properties (18). Functional and structural assays revealed that D553N expresses little ionic currents, which is possibly due to the defective channel trafficking so that the channels cannot reach the plasma membrane for normal functions (18).The Src kinase family has nine members (19). They are closely related and share the same regulatory function. Three of them, Src, Fyn, and Yes, are ubiquitously expressed in a variety of tissues including neurons and myocytes (19, 20). Without stimulation, they are inactive. However, mutation of key tyrosine residue results in the constitutively active form of the kinase, SrcY529F, FynY531F, and YesY537F, respectively (15, 21, 22). Using these Src kinases, we show in this report a novel approach that can restore the surface expression of D553N for normal current expression via tyrosine phosphorylation.     

Phosphoproteome Analysis of Functional Mitochondria Isolated from Resting Human Muscle Reveals Extensive Phosphorylation of Inner Membrane Protein Complexes and Enzymes

Mitochondria play a central role in energy metabolism and cellular survival, and consequently mitochondrial dysfunction is associated with a number of human pathologies. Reversible protein phosphorylation emerges as a central mechanism in the regulation of several mitochondrial processes. In skeletal muscle, mitochondrial dysfunction is linked to insulin resistance in humans with obesity and type 2 diabetes. We performed a phosphoproteomics study of functional mitochondria isolated from human muscle biopsies with the aim to obtain a comprehensive overview of mitochondrial phosphoproteins. Combining an efficient mitochondrial isolation protocol with several different phosphopeptide enrichment techniques and LC-MS/MS, we identified 155 distinct phosphorylation sites in 77 mitochondrial phosphoproteins, including 116 phosphoserine, 23 phosphothreonine, and 16 phosphotyrosine residues. The relatively high number of phosphotyrosine residues suggests an important role for tyrosine phosphorylation in mitochondrial signaling. Many of the mitochondrial phosphoproteins are involved in oxidative phosphorylation, tricarboxylic acid cycle, and lipid metabolism, i.e. processes proposed to be involved in insulin resistance. We also assigned phosphorylation sites in mitochondrial proteins involved in amino acid degradation, importers and transporters, calcium homeostasis, and apoptosis. Bioinformatics analysis of kinase motifs revealed that many of these mitochondrial phosphoproteins are substrates for protein kinase A, protein kinase C, casein kinase II, and DNA-dependent protein kinase. Our results demonstrate the feasibility of performing phosphoproteome analysis of organelles isolated from human tissue and provide novel targets for functional studies of reversible phosphorylation in mitochondria. Future comparative phosphoproteome analysis of mitochondria from healthy and diseased individuals will provide insights into the role of abnormal phosphorylation in pathologies, such as type 2 diabetes.Mitochondria are the primary energy-generating systems in eukaryotes. They play a crucial role in oxidative metabolism, including carbohydrate metabolism, fatty acid oxidation, and urea cycle, as well as in calcium signaling and apoptosis (1, 2). Mitochondrial dysfunction is centrally involved in a number of human pathologies, such as type 2 diabetes, Parkinson disease, and cancer (3). The most prevalent form of cellular protein post-translational modifications (PTMs),1 reversible phosphorylation (4–6), is emerging as a central mechanism in the regulation of mitochondrial functions (7, 8). The steadily increasing numbers of reported mitochondrial kinases, phosphatases, and phosphoproteins imply an important role of protein phosphorylation in different mitochondrial processes (9–11).Mass spectrometry (MS)-based proteome analysis is a powerful tool for global profiling of proteins and their PTMs, including protein phosphorylation (12, 13). A variety of proteomics techniques have been developed for specific enrichment of phosphorylated proteins and peptides and for phosphopeptide-specific data acquisition techniques at the MS level (14). Enrichment methods based on affinity chromatography, such as titanium dioxide (TiO₂) (15–17), zwitterionic hydrophilic interaction chromatography (ZIC-HILIC) (18), immobilized metal affinity chromatography (IMAC) (19, 20), and ion exchange chromatography (strong anion exchange and strong cation exchange) (21, 22), have shown high efficiencies for enrichment of phosphopeptides (14). Recently, we demonstrated that calcium phosphate precipitation (CPP) is highly effective for enriching phosphopeptides (23). It is now generally accepted that no single method is comprehensive, but combinations of different enrichment methods produce distinct overlapping phosphopeptide data sets to enhance the overall results in phosphoproteome analysis (24, 25). Phosphopeptide sequencing by mass spectrometry has seen tremendous advances during the last decade (26). For example, MS/MS product ion scanning, multistage activation, and precursor ion scanning are effective methods for identifying serine (Ser), threonine (Thr), and tyrosine (Tyr) phosphorylated peptides (14, 26).A “complete” mammalian mitochondrial proteome was reported by Mootha and co-workers (27) and included 1098 proteins. The mitochondrial phosphoproteome has been characterized in a series of studies, including yeast, mouse and rat liver, porcine heart, and plants (19, 28–31). To date, the largest data set by Deng et al. (30) identified 228 different phosphoproteins and 447 phosphorylation sites in rat liver mitochondria. However, the in vivo phosphoproteome of human mitochondria has not been determined. A comprehensive mitochondrial phosphoproteome is warranted for further elucidation of the largely unknown mechanisms by which protein phosphorylation modulates diverse mitochondrial functions.The percutaneous muscle biopsy technique is an important tool in the diagnosis and management of human muscle disorders and has been widely used to investigate metabolism and various cellular and molecular processes in normal and abnormal human muscle, in particular the molecular mechanism underlying insulin resistance in obesity and type 2 diabetes (32). Skeletal muscle is rich in mitochondria and hence a good source for a comprehensive proteomics and functional analysis of mitochondria (32, 33).The major aim of the present study was to obtain a comprehensive overview of site-specific phosphorylation of mitochondrial proteins in functionally intact mitochondria isolated from human skeletal muscle. Combining an efficient protocol for isolation of skeletal muscle mitochondria with several different state-of-the-art phosphopeptide enrichment methods and high performance LC-MS/MS, we identified 155 distinct phosphorylation sites in 77 mitochondrial phosphoproteins, many of which have not been reported before. We characterized this mitochondrial phosphoproteome by using bioinformatics tools to classify functional groups and functions, including kinase substrate motifs.