Quantitative Measurement of in Vivo Phosphorylation States of Cdk5 Activator p35 by Phos-tag SDS-PAGE

Phosphorylation is a major post-translational modification widely used in the regulation of many cellular processes. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase activated by activation subunit p35. Cdk5-p35 regulates various neuronal activities such as neuronal migration, spine formation, synaptic activity, and cell death. The kinase activity of Cdk5 is regulated by proteolysis of p35: proteasomal degradation causes down-regulation of Cdk5, whereas cleavage of p35 by calpain causes overactivation of Cdk5. Phosphorylation of p35 determines the proteolytic pathway. We have previously identified Ser⁸ and Thr¹³⁸ as major phosphorylation sites using metabolic labeling of cultured cells followed by two-dimensional phosphopeptide mapping and phosphospecific antibodies. However, these approaches cannot determine the extent of p35 phosphorylation in vivo. Here we report the use of Phos-tag SDS-PAGE to reveal the phosphorylation states of p35 in neuronal culture and brain. Using Phos-tag acrylamide, the electrophoretic mobility of phosphorylated p35 was delayed because it is trapped at Phos-tag sites. We found a novel phosphorylation site at Ser⁹¹, which was phosphorylated by Ca²⁺-calmodulin-dependent protein kinase II in vitro. We constructed phosphorylation-dependent banding profiles of p35 and Ala substitution mutants at phosphorylation sites co-expressed with Cdk5 in COS-7 cells. Using the standard banding profiles, we assigned respective bands of endogenous p35 with combinations of phosphorylation states and quantified Ser⁸, Ser⁹¹, and Thr¹³⁸ phosphorylation. The highest level of p35 phosphorylation was observed in embryonic brain; Ser⁸ was phosphorylated in all p35 molecules, whereas Ser⁹¹ was phosphorylated in 60% and Thr¹³⁸ was phosphorylated in ∼12% of p35 molecules. These are the first quantitative and site-specific measurements of phosphorylation of p35, demonstrating the usefulness of Phos-tag SDS-PAGE for analysis of phosphorylation states of in vivo proteins.Phosphorylation is a major post-translational modification of proteins, modulating a variety of cellular functions (1, 2). Because most phosphorylation occurs in a highly site-specific manner, identification of phosphorylation sites has been a subject of intense investigation. Several analytical methods have been utilized to identify phosphorylation sites, including mass spectrometry, amino acid sequencing, and radioisotope phosphate labeling of proteins with mutation(s) at putative phosphorylation site(s) (3, 4). Phosphorylation site-specific antibodies are frequently used to detect phosphorylation at target sites (5, 6). Many phosphospecific antibodies are now commercially available. These phosphospecific antibodies are convenient and useful tools for examining site-specific phosphorylation both in vivo and in vitro. However, they are not appropriate for estimating quantitative ratios of phosphorylation states. Electrophoretic mobility shift on SDS-PAGE is also often used to observe phosphorylation (7–10), but this method is not always applied to site-specific phosphorylation.Phos-tag is a newly developed dinuclear metal complex that can be used to provide phosphate-binding sites when conjugated to analytical materials such as acrylamide and biotin (11). In SDS-PAGE using Phos-tag acrylamide, phosphorylated proteins are trapped by the Phos-tag sites, delaying their migration and thus separating them from unphosphorylated proteins. Subsequent immunoblot analysis with phosphorylation-independent antibodies reveals both the phosphorylated and unphosphorylated bands. Because the migration of the phosphorylated proteins is greatly delayed compared with migration in Laemmli SDS-PAGE, it is easy to identify the phosphorylated proteins from observed positions on blots. In the past 3 years, this method has been used to detect phosphorylation states for many proteins such as ERK1/2, cdc37, myosin light chain, eIF2α, protein kinase D, β-casein, SIRT7, and dysbindin-1 (12–21).Cyclin-dependent kinase 5 (Cdk5)¹ is a proline-directed serine/threonine kinase that is expressed predominantly in postmitotic neurons and regulates various neuronal events such as neuronal migration, spine formation, synaptic activity, and cell death (22–24). Cdk5 is activated by binding to activation subunit p35 and inactivated by proteasomal degradation of p35 (25). In addition, Cdk5 activity is deregulated by cleavage of p35 to p25 with calpain, resulting in abnormal activation and ultimately causing neuronal cell death (26–29). Proteolysis of p35, either by proteasomal degradation or cleavage by calpain, is regulated by phosphorylation of p35 by Cdk5 (30–33). Therefore, phosphorylation of p35 is essential for proper regulation of Cdk5 activity and function. We previously identified Ser⁸ and Thr¹³⁸ as major p35 phosphorylation sites (33). We also showed that phosphorylation of p35 decreased during brain development and proposed its relationship to age-dependent vulnerability of neurons to stress stimuli (32). Thus, to understand the in vivo regulation of Cdk5 activity, it is critical to analyze the phosphorylation states of p35 in brain. However, there is no convenient method to analyze the precise in vivo phosphorylation status of the endogenous proteins.In this study, we applied the Phos-tag SDS-PAGE method to analyze the phosphorylation states of p35 in vivo and in cultured neurons. We constructed standard band profiles of phosphorylated p35 by Phos-tag SDS-PAGE using Ala mutants at Ser⁸ and/or Thr¹³⁸. From these experiments, we observed an unidentified in vivo phosphorylation site at Ser⁹¹. We quantified the phosphorylation at each site in cultured neurons and brain, providing the first quantitative estimate of the in vivo phosphorylation states of p35. We discuss the usefulness of Phos-tag SDS-PAGE to analyze the in vivo phosphorylation states of proteins.     

Arabidopsis LIP5, a Positive Regulator of Multivesicular Body Biogenesis,Is a Critical Target of Pathogen-Responsive MAPK Cascade in Plant Basal Defense

Multivesicular bodies (MVBs) play essential roles in many cellular processes. The MVB pathway requires reversible membrane association of the endosomal sorting complexes required for transports (ESCRTs) for sustained protein trafficking. Membrane dissociation of ESCRTs is catalyzed by the AAA ATPase SKD1, which is stimulated by LYST-INTERACTING PROTEIN 5 (LIP5). We report here that LIP5 is a target of pathogen-responsive mitogen-activated protein kinases (MPKs) and plays a critical role in plant basal resistance. Arabidopsis LIP5 interacts with MPK6 and MPK3 and is phosphorylated in vitro by activated MPK3 and MPK6 and in vivo upon expression of MPK3/6-activating NtMEK2^DD and pathogen infection. Disruption of LIP5 has little effects on flg22-, salicylic acid-induced defense responses but compromises basal resistance to Pseudomonas syringae. The critical role of LIP5 in plant basal resistance is dependent on its ability to interact with SKD1. Mutation of MPK phosphorylation sites in LIP5 does not affect interaction with SKD1 but reduces the stability and compromises the ability to complement the lip5 mutant phenotypes. Using the membrane-selective FM1–43 dye and transmission electron microscopy, we demonstrated that pathogen infection increases formation of both intracellular MVBs and exosome-like paramural vesicles situated between the plasma membrane and the cell wall in a largely LIP5-dependent manner. These results indicate that the MVB pathway is positively regulated by pathogen-responsive MPK3/6 through LIP5 phosphorylation and plays a critical role in plant immune system likely through relocalization of defense-related molecules.     

CAPS Activity in Priming Vesicle Exocytosis Requires CK2 Phosphorylation

CAPS (Ca²⁺-dependent activator protein for secretion) functions in priming Ca²⁺-dependent vesicle exocytosis, but the regulation of CAPS activity has not been characterized. Here we show that phosphorylation by protein kinase CK2 is required for CAPS activity. Dephosphorylation eliminated CAPS activity in reconstituting Ca²⁺-dependent vesicle exocytosis in permeable and intact PC12 cells. Ser-5, -6, and -7 and Ser-1281 were identified by mass spectrometry as the major phosphorylation sites in the 1289 residue protein. Ser-5, -6, and -7 but not Ser-1281 to Ala substitutions abolished CAPS activity. Protein kinase CK2 phosphorylated CAPS in vitro at these sites and restored the activity of dephosphorylated CAPS. CK2 is the likely in vivo CAPS protein kinase based on inhibition of phosphorylation by tetrabromo-2-benzotriazole in PC12 cells and by the identity of in vivo and in vitro phosphorylation sites. CAPS phosphorylation by CK2 was constitutive, but the elevation of Ca²⁺ in synaptosomes increased CAPS Ser-5 and -6 dephosphorylation, which terminates CAPS activity. These results identify a functionally important N-terminal phosphorylation site that regulates CAPS activity in priming vesicle exocytosis.Regulated neurotransmitter secretion is central to intercellular communication in the nervous system. Two types of secretory vesicles mediate neurotransmitter release; that is, synaptic vesicles that release transmitters such as glutamate at synapses and dense-core vesicles that release modulatory transmitters and neuropeptides at non-synaptic sites. Both types of secretory vesicles are recruited to docking sites on the plasma membrane where they are primed to a ready release state to undergo fusion in response to Ca²⁺ elevations. Many of the proteins that mediate the targeting, docking, priming, and Ca²⁺-dependent fusion of vesicles with the plasma membrane function in both synaptic vesicle and dense-core vesicle pathways (1). CAPS-1² (also known as Cadps1) is a 1289-residue protein that reconstitutes Ca²⁺-triggered dense-core vesicle exocytosis in permeable neuroendocrine cells at a priming step (2–4). CAPS is required for secretion of a subset of transmitters in Caenorhabditis elegans (5) and Drosophila melanogaster (6) and for priming dense-core vesicle exocytosis in neuroendocrine cells (7) and synaptic vesicle exocytosis in neurons (8). Vesicle priming reactions are extensively modulated during physiological demand (9), but mechanisms that regulate CAPS function remain to be identified.Reversible protein phosphorylation is a major mechanism for the regulation of cellular processes including vesicle exocytosis. Many proteins that function in evoked vesicle exocytosis are phosphoproteins (10, 11). The neuronal SNARE proteins syntaxin 1A, VAMP-2, and SNAP-25 are phosphorylated by several protein kinases in vitro (12–14). Protein kinase C and protein kinase A sites on SNAP-25 affect refilling rates and size, respectively, of the primed pool of vesicles in chromaffin cells (15, 16). Several SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-binding proteins such as munc18, RIM1, and rabphilin undergo regulated phosphorylation, but it is not known whether phosphorylation affects function (10, 11, 17).Because the function of CAPS at a priming step in vesicle exocytosis may be regulated, we determined whether CAPS is phosphorylated. We show that CAPS is a phosphoprotein with functionally essential N-terminal phosphorylated Ser residues. Ser-5, -6, and -7 in CAPS were substrates for protein kinase CK2 in vitro and in vivo as well as for a Ca²⁺-dependent dephosphorylation mechanism. The results indicate that phosphorylation by protein kinase CK2 is necessary for CAPS activity in priming vesicle exocytosis and that regulated dephosphorylation may constitute a mechanism for terminating CAPS activity.     

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.     

Large-scale Proteomics Analysis of the Human Kinome

Members of the human protein kinase superfamily are the major regulatory enzymes involved in the activity control of eukaryotic signal transduction pathways. As protein kinases reside at the nodes of phosphorylation-based signal transmission, comprehensive analysis of their cellular expression and site-specific phosphorylation can provide important insights into the architecture and functionality of signaling networks. However, in global proteome studies, low cellular abundance of protein kinases often results in rather minor peptide species that are occluded by a vast excess of peptides from other cellular proteins. These analytical limitations create a rationale for kinome-wide enrichment of protein kinases prior to mass spectrometry analysis. Here, we employed stable isotope labeling by amino acids in cell culture (SILAC) to compare the binding characteristics of three kinase-selective affinity resins by quantitative mass spectrometry. The evaluated pre-fractionation tools possessed pyrido[2,3-d]pyrimidine-based kinase inhibitors as immobilized capture ligands and retained considerable subsets of the human kinome. Based on these results, an affinity resin displaying the broadly selective kinase ligand VI16832 was employed to quantify the relative expression of more than 170 protein kinases across three different, SILAC-encoded cancer cell lines. These experiments demonstrated the feasibility of comparative kinome profiling in a compact experimental format. Interestingly, we found high levels of cytoplasmic and low levels of receptor tyrosine kinases in MV4–11 leukemia cells compared with the adherent cancer lines HCT116 and MDA-MB-435S. The VI16832 resin was further exploited to pre-fractionate kinases for targeted phosphoproteomics analysis, which revealed about 1200 distinct phosphorylation sites on more than 200 protein kinases. This hitherto largest survey of site-specific phosphorylation across the kinome significantly expands the basis for functional follow-up studies on protein kinase regulation. In conclusion, the straightforward experimental procedures described here enable different implementations of kinase-selective proteomics with considerable potential for future signal transduction and kinase drug target analysis.Reversible protein phosphorylation represents the most common type of post-translational modification (PTM)¹ in eukaryotic organisms. A plethora of studies on a large variety of proteins have established that site-specific phosphorylation events fulfill key functions in the activity control of signaling cascades and networks (1). Cellular protein phosphorylation is controlled by more than 500 members of the protein kinase superfamily, which comprises one of the largest enzyme families encoded by the human genome (2). Protein kinases represent the key elements in phosphorylation-based signal transmission. Aberrant protein kinase expression and/or activity, often because of gene amplification or mutational changes, is involved in pathological processes leading to malignant transformation and tumor development (3). Therefore, protein kinases have emerged as a major class of drug targets for therapeutic intervention (4–6). Given the diversity of molecular mechanisms related to de-regulated kinase function in human cancers, proteomic approaches could significantly enhance our understanding of disease-relevant kinase function and also help to optimize and adjust therapeutic strategies. In addition to assessing protein expression, the analysis of site-specific phosphorylations on protein kinases is of particular relevance, as these PTMs can be indicative of their cellular catalytic activities (7, 8). Protein kinases can not only modulate each other''s functions and activities through site-specific phosphorylation events, but often also undergo site-specific autophosphorylation once they get activated (9). Thus, the comprehensive assessment of kinase-derived phosphopeptides can provide important insights into the regulation of these key players in phosphorylation-controlled signaling.Regulatory enzymes such as protein kinases are often expressed at low cellular levels. This can impede their detection by LC-MS in highly complex peptide mixtures derived from total cell or tissue extracts. These analytical challenges are further aggravated in phosphoproteomic experiments due to the fact that many phosphopeptide species result from sub-stoichiometric phosphorylation events (10). Consequently, phosphopeptide isolation methods have proven to be essential. Among others, techniques such as immobilized metal affinity chromatography or enrichment by means of titanium dioxide (TiO₂)-coated beads have found widespread use in MS-based phosphoproteomics (11–13). In addition, to reduce initial sample complexity, either protein fractionation by gel electrophoresis or peptide separation by strong cation exchange chromatography is typically included in contemporary phosphoproteomics workflows (14–16). These separation techniques in combination with LC-MS on state-of-the-art mass spectrometers enabled the identification of thousands of phosphorylation sites from total cellular extracts (15, 17, 18). Despite these impressive advances, such large-scale efforts require considerable instrument time, and the current methodology is still not comprehensive across the full dynamic range of the entire phosphoproteome. This creates the rationale for sub-proteome analyses to achieve high coverage and analytical sensitivity, which is particularly relevant for members of the protein kinase enzyme family.To date, the only pre-fractionation techniques permitting the enrichment of more than a few protein kinases are affinity capture methods relying on immobilized and kinase-selective small molecule inhibitors (19–21). We and others have demonstrated that combinations of such kinase inhibitor resins efficiently pre-fractionate kinases for subsequent phosphorylation analysis (7, 22, 23). Ideally, capture molecules for kinase proteomics have two properties. First, they should exhibit high non-selectivity within the kinase superfamily. Second, they should efficiently discriminate between protein kinases and other classes of cellular proteins under the biochemical conditions of the pre-fractionation procedure.In our efforts to characterize affinity reagents fulfilling these criteria, we quantitatively compared a selection of immobilized pyrido[2,3-d]pyrimidine-based inhibitors with respect to their proteome-wide kinase binding properties. Based on this assessment, an affinity matrix displaying the small molecule VI16832 was used as an enrichment tool for the comparative expression analysis of protein kinases in different cancer cell lines. The highly efficient VI16832 affinity resin further enabled a large-scale phosphoproteomics survey resulting in the identification and confident assignment of about 1200 phosphorylation sites on more than 200 distinct protein kinases.     

Identification of Direct Tyrosine Kinase Substrates Based on Protein Kinase Assay-Linked Phosphoproteomics

Protein kinases are implicated in multiple diseases such as cancer, diabetes, cardiovascular diseases, and central nervous system disorders. Identification of kinase substrates is critical to dissecting signaling pathways and to understanding disease pathologies. However, methods and techniques used to identify bona fide kinase substrates have remained elusive. Here we describe a proteomic strategy suitable for identifying kinase specificity and direct substrates in high throughput. This approach includes an in vitro kinase assay-based substrate screening and an endogenous kinase dependent phosphorylation profiling. In the in vitro kinase reaction route, a pool of formerly phosphorylated proteins is directly extracted from whole cell extracts, dephosphorylated by phosphatase treatment, after which the kinase of interest is added. Quantitative proteomics identifies the rephosphorylated proteins as direct substrates in vitro. In parallel, the in vivo quantitative phosphoproteomics is performed in which cells are treated with or without the kinase inhibitor. Together, proteins phosphorylated in vitro overlapping with the kinase-dependent phosphoproteome in vivo represents the physiological direct substrates in high confidence. The protein kinase assay-linked phosphoproteomics was applied to identify 25 candidate substrates of the protein-tyrosine kinase SYK, including a number of known substrates and many novel substrates in human B cells. These shed light on possible new roles for SYK in multiple important signaling pathways. The results demonstrate that this integrated proteomic approach can provide an efficient strategy to screen direct substrates for protein tyrosine kinases.Protein phosphorylation plays a pivotal role in regulating biological events such as protein–protein interactions, signal transduction, subcellular localization, and apoptosis (1). Deregulation of kinase-substrate interactions often leads to disease states such as human malignancies, diabetes, and immune disorders (2). Although a number of kinases are being targeted to develop new drugs, our understanding of the precise relationships between protein kinases and their direct substrates is incomplete for the majority of protein kinases (3). Thus, mapping kinase–substrate relationships is essential for the understanding of biological signaling networks and the discovery and development of drugs for targeted therapies (4). Toward this goal, various in vitro kinase assays using synthetic peptide libraries (5), phage expression libraries (6), protein arrays (7–9), or cell extracts (10, 11) have been explored for the screening of kinase substrates.Besides classical biochemical and genetic methods, mass spectrometry-based high throughput approaches have become increasingly attractive because they are capable of sequencing proteins and localizing phosphorylation sites at the same time. Mass spectrometry-based proteomic methods have been extensively applied to kinase-substrate interaction mapping (12) and global phosphorylation profiling (13–15). Although thousands of phosphorylation events can be inspected simultaneously (16, 17), large-scale phosphoproteomics does not typically reveal direct relationships between protein kinases and their substrates.Recently, several mass spectrometry-based proteomic strategies have been introduced for identifying elusive kinase substrates (7, 18, 19). Taking advantage of recent advances of high speed and high-resolution mass spectrometry, these methods used purified, active kinases to phosphorylate cell extracts in vitro, followed by mass spectrometric analysis to identify phosphoproteins. These approaches commonly face the major challenge of distinguishing phosphorylation events triggered by the kinase reaction from background signals introduced by endogenous kinase activities (20). To dissect the phosphorylation cascade, Shokat and colleagues developed an approach named Analog-Sensitive Kinase Allele (ASKA)¹ (21). In their approach, a kinase is engineered to accept a bulky-ATP analog exclusively so that direct phosphorylation caused by the analog-sensitive target kinase can be differentiated from that of wild type kinases. As a result, indirect effects caused by contaminating kinases during the in vitro kinase assay are largely eliminated. ASKA has recently been coupled with quantitative proteomics, termed Quantitative Identification of Kinase Substrates (QIKS) (12), to identify substrate proteins of Mek1. Recently, one extension of the ASKA technique is for the analog ATP to carry a γ-thiophosphate group so that in vitro thiophosphorylated proteins can be isolated for mass spectrometric detection (22–24). In addition to ASKA, radioisotope labeling using [γ-³²P]ATP (10), using concentrated purified kinase (25), inactivating endogenous kinase activity by an additional heating step (11), and quantitative proteomics (26, 27) are alternative means aimed to address the same issues. All of these methods, however, have been limited to the identification of in vitro kinase substrates.To bridge the gap between in vitro phosphorylation and physiological phosphorylation events, we have recently introduced an integrated strategy termed Kinase Assay-Linked Phosphoproteomics (KALIP) (28). By combining in vitro kinase assays with in vivo phosphoproteomics, this method was demonstrated to have exceptional sensitivity for high confidence identification of direct kinase substrates. The main drawback for the KALIP approach is that the kinase reaction is performed at the peptide stage to eliminate any problems related to contamination by endogenous kinases. However, the KALIP method may not be effective for kinases that require a priming phosphorylation event (i.e. a previous phosphorylation, on substrate or kinase, has effect on following phosphorylation) (29), additional interacting surfaces (30), or a docking site on the protein (31). For example, basophilic kinases require multiple basic resides for phosphorylation and tryptic digestion will abolish these motifs, which are needed for effective kinase reactions.We address the shortcoming by introducing an alternative strategy termed Protein Kinase Assay-Linked Phosphoproteomics (proKALIP). The major difference between this method and the previous KALIP method is the utilization of protein extracts instead of digested peptides as the substrate pool. The major issue is how to reduce potential interference by endogenous kinase activities. One effective solution is to use a generic kinase inhibitor, 5′-(4-fluorosulfonylbenzoyl)adenosine (FSBA), which was widely used for covalent labeling of kinases (32, 33), kinase isolation (34), kinase activity exploration (35, 36), and more recently kinase substrate identification by Kothary and co-workers (37). However, an extra step is required to effectively remove the inhibitor before the kinase reaction, which may decrease the sensitivity. ProKALIP addresses the issue by carrying out the kinase reaction using formerly in vivo phosphorylated proteins as candidates. This step efficiently improves the sensitivity and specificity of the in vitro kinase reaction. Coupled with in vivo phosphoproteomics, proKALIP has gained a high sensitivity and provided physiologically relevant substrates with high confidence.To demonstrate the proKALIP strategy, the protein-tyrosine kinase SYK was used as our target kinase. SYK is known to play a crucial role in the adaptive immune response, particularly in B cells, by facilitating the antigen induced B-cell receptor (BCR) signaling pathways and modulating cellular responses to oxidative stress in a receptor-independent manner (38, 39). SYK also has diverse biological functions such as innate immune recognition, osteoclast maturation, cellular adhesion, platelet activation, and vascular development (38). In addition, the expression of SYK is highly correlated to tumorigenesis by promoting cell–cell adhesion and inhibiting the motility, growth, and invasiveness of certain cancer cells (40). In this study, we attempt to identify bona fide substrates of SYK in human B cells using the proKALIP approach and demonstrate the specificity and sensitivity of this strategy.     

Identification of additional MAP kinases activated upon PAMP treatment

Mitogen-activated protein (MAP) kinase cascades play important roles in plant immunity. Upon pathogen associated molecular pattern (PAMP) treatment, MPK3, MPK6 and MPK4 are quickly activated by upstream MKKs through phosphorylation. Western blot analysis using α-phospho-p44/42-ERK antibody suggests that additional MPKs with similar size as MPK4 are also activated upon PAMP perception. To identify these MAP kinases, 7 candidate MPKs with similar sizes as MPK4 were selected for further analysis. Transgenic plants expressing these MPKs with a ZZ-3xFLAG double tag of 17 kD were generated and analyzed by western blot. MPK1, MPK11 and MPK13 were found to be phosphorylated upon treatment with flg22. Our study revealed additional MAPKs being activated during PAMP-triggered immunity.     

Systems-wide Analysis of a Phosphatase Knock-down by Quantitative Proteomics and Phosphoproteomics

Signal transduction in metazoans regulates almost all aspects of biological function, and aberrant signaling is involved in many diseases. Perturbations in phosphorylation-based signaling networks are typically studied in a hypothesis-driven approach, using phospho-specific antibodies. Here we apply quantitative, high-resolution mass spectrometry to determine the systems response to the depletion of one signaling component. Drosophila cells were metabolically labeled using stable isotope labeling by amino acids in cell culture (SILAC) and the phosphatase Ptp61F, the ortholog of mammalian PTB1B, a drug target for diabetes, was knocked down by RNAi. In total we detected more than 10,000 phosphorylation sites in the phosphoproteome of Drosophila Schneider cells and trained a phosphorylation site predictor with this data. SILAC-based quantitation after phosphatase knock-down showed that apart from the phosphatase, the proteome was minimally affected whereas 288 of 6,478 high-confidence phosphorylation sites changed significantly. Responses at the phosphotyrosine level included the already described Ptp61F substrates Stat92E and Abi. Our analysis highlights a connection of Ptp61F to cytoskeletal regulation through GTPase regulating proteins and focal adhesion components.Information processing in biological systems relies heavily on activation and inactivation of proteins by phosphorylation. This key post-translational modification is involved in the regulation of most cellular processes and mediates many rapid responses as well as long-term gene expression changes in response to stimuli. Protein kinases and protein phosphatases coordinately regulate this highly dynamic and reversible modification. Phosphorylation is usually studied in a candidate-based approach by in vitro kinase assays or by immune techniques employing phospho-specific antibodies. Despite the success of this reductionist approach, it does not afford a systems-wide observation of the effects upon perturbations of signaling networks.Recent advances in MS-based¹ proteomics now allow the identification of thousands of phosphorylation sites from complex protein mixtures (1–3). Most large-scale phosphoproteomics studies have been qualitative rather than quantitative; however, isotope-based methods enable precise quantitation of phosphorylation sites between two or more cellular states (4–6). Our group has applied the metabolic labeling technology termed stable isotope labeling by amino acids in cell culture (SILAC) (7) for the quantitative comparison of phosphoproteomes. For example, we quantified phosphorylation dynamics in response to epidermal growth factor stimulation. Out of a measured phosphoproteome of several thousand sites only a minority (about 10%) was regulated by the signal, highlighting the importance of quantitation in pinpointing specific systems responses (8).Drosophila is a well established model system to study key players in cell signaling and development. Genetic studies have been performed for decades whereas more recently also RNA interference (RNAi) has been employed for gene function studies using a highly efficient silencing protocol (9). A further advantage of Drosophila as a model system is the lower degree of functional redundancy compared with higher vertebrates while maintaining a high level of conservation of human genes linked to disease (10).Two large-scale, non-quantitative Drosophila phosphoproteome studies were carried out in embryonic Kc167 cells (11) and embryos (12). Both studies identified more than 10,000 sites of the Drosophila phosphoproteome.We have recently adapted the SILAC methodology for quantitative proteomics to Drosophila. Schneider line 2 (SL2) cells were treated with either mock dsRNA or dsRNA against ISWI, a component of chromatin remodeling complexes. The combination of RNAi and SILAC allows the unbiased “phenotypization” of the gene knock-down directly at the proteome level (13).Here we determined a high-quality basal phosphoproteome in SL2 cells and characterized its structural and evolutionary properties. We compared kinase substrate motives between Drosophila and human and trained a Drosophila phosphorylation site predictor.To explore the potential of quantitative phosphoproteomics in a systems-wide manner, we focused on the Drosophila non-transmembrane tyrosine phosphatase Ptp61F. This phosphatase is the ortholog of mammalian PTB1B, which is thought to be involved in type 2 diabetes, obesity, and cancer (14), and which is the target of several ongoing drug development projects (15). Ptp61F is a negative regulator of JAK/STAT signaling (16, 17) and, together with the Ableson kinase (Abl), involved in the regulation of the Abl interacting protein (Abi) and lamella formation (18). Both PTP1B and Ptp61F are among the best studied protein tyrosine phosphatases in their respective organisms; however the characterization of their substrates is still far from complete. Two recent mass spectrometric studies employed substrate trapping to identify direct substrates of PTP1B and Ptp61F (19, 20). The PTP1B study was combined with phosphotyrosine peptide enrichment, which led to site-specific detection of potential PTP1B targets. PTP1B function was additionally investigated by quantitative phosphotyrosine proteomics comparing wild type and PTP1B-deficient fibroblasts. In contrast, the Ptp61F study identified potential substrates without site-specific information. One of these was PVR, the Drosophila homolog of VEGFR and PDGFR, suggesting that Ptp61F - like its mammalian counterpart - counteracts receptor tyrosine kinase signaling. Apart from Abi, further components of the SCAR/WAVE complex as well as its regulatory kinase Abl were identified as potential Ptp61F substrates. This supports an involvement of Ptp61F in the regulation of actin reorganization and remodeling.To study the role of Ptp61F in a global and unbiased approach we combined global quantitative phosphoproteome analysis with RNA interference. We profiled tyrosine, serine and threonine phosphorylation changes upon ablation of Ptp61F by RNAi. In parallel, we quantified changes in the proteome, which allowed us to normalize changes in phosphorylation sites to corresponding changes at the protein level. Interestingly, we observed increased tyrosine phosphorylation of the protein tyrosine kinase Abl which suggests an enhanced Abl activity upon Ptp61F RNAi. We additionally detected up-regulated phosphotyrosine sites on GTPase regulating proteins (like RhoGAP15B and Vav) and constituents of focal adhesions (like Paxillin and Lasp) which expand the proposed involvement of Ptp61F in the regulation of cytoskeleton organization. Our work represents proof-of-principle that the combination of large-scale phosphoproteomics and a loss-of-function approach can contribute significantly to elucidating the role of key players in phosphorylation-dependent signaling. Importantly, this systems-wide approach measures the net effect of the perturbation on the entire signaling network, without the need to define specific substrate-kinase or substrate -phosphatase relationships or other direct functional mechanisms.     

O-Linked N-Acetylglucosamine Modification of Insulin Receptor Substrate-1 Occurs in Close Proximity to Multiple SH2 Domain Binding Motifs

Insulin receptor substrate-1 (IRS-1) is a highly phosphorylated adaptor protein critical to insulin and IGF-1 receptor signaling. Ser/Thr kinases impact the metabolic and mitogenic effects elicited by insulin and IGF-1 through feedback and feed forward regulation at the level of IRS-1. Ser/Thr residues of IRS-1 are also O-GlcNAc-modified, which may influence the phosphorylation status of the protein. To facilitate the understanding of the functional effects of O-GlcNAc modification on IRS-1-mediated signaling, we identified the sites of O-GlcNAc modification of rat and human IRS-1. Tandem mass spectrometric analysis of IRS-1, exogenously expressed in HEK293 cells, revealed that the C terminus, which is rich in docking sites for SH2 domain-containing proteins, was O-GlcNAc-modified at multiple residues. Rat IRS-1 was O-GlcNAc-modified at Ser⁹¹⁴, Ser¹⁰⁰⁹, Ser¹⁰³⁶, and Ser¹⁰⁴¹. Human IRS-1 was O-GlcNAc-modified at Ser⁹⁸⁴ or Ser⁹⁸⁵, at Ser¹⁰¹¹, and possibly at multiple sites within residues 1025–1045. O-GlcNAc modification at a conserved residue in rat (Ser¹⁰⁰⁹) and human (Ser¹⁰¹¹) IRS-1 is adjacent to a putative binding motif for the N-terminal SH2 domains of p85α and p85β regulatory subunits of phosphatidylinositol 3-kinase and the tyrosine phosphatase SHP2 (PTPN11). Immunoblot analysis using an antibody generated against human IRS-1 Ser¹⁰¹¹ GlcNAc further confirmed the site of attachment and the identity of the +203.2-Da mass shift as β-N-acetylglucosamine. The accumulation of IRS-1 Ser¹⁰¹¹ GlcNAc in HEPG2 liver cells and MC3T3-E1 preosteoblasts upon inhibition of O-GlcNAcase indicates that O-GlcNAcylation of endogenously expressed IRS-1 is a dynamic process that occurs at normal glucose concentrations (5 mm). O-GlcNAc modification did not occur at any known or newly identified Ser/Thr phosphorylation sites and in most cases occurred simultaneously with phosphorylation of nearby residues. These findings suggest that O-GlcNAc modification represents an additional layer of posttranslational regulation that may impact the specificity of effects elicited by insulin and IGF-1.Insulin receptor substrate-1 (IRS-1)1 is a highly phosphorylated adaptor protein critical to insulin and IGF-1 receptor signaling. Many of the metabolic and mitogenic effects elicited by insulin and IGF-1 are mediated and modulated by posttranslational modifications of IRS-1, and tight regulation at the posttranslational level is crucial for maintaining insulin sensitivity and controlling growth factor-induced proliferation. Following hormonal stimulation, IRS-1 is phosphorylated by the receptor tyrosine kinases creating SH2 domain docking sites for downstream binding partners including the p85 regulatory subunits of phosphatidylinositol 3-kinase, Grb2, and the tyrosine phosphatase SHP2 (PTPN11) (1). Binding of p85 phosphatidylinositol 3-kinase and Grb2 activate the PI3K/Akt and Ras-MAPK pathways, respectively, whereas binding of SHP2 results in tyrosine dephosphorylation and signal attenuation (2). Positive and negative feedback regulation by Ser/Thr kinases, such as Akt (3), c-Jun N-terminal kinase (JNK) (4), S6K (5), and ERK (6), impact the interactions of IRS-1 with SH2 domain proteins and the receptor thereby affecting the duration and outcome of the signal. IRS-1 has been described as a central node for the integration of information regarding the nutrient and stress status of the cell (7). This information is encoded by site-specific phosphorylation by a number of kinases that regulate the specificity of effects that are elicited following receptor stimulation. Many sites of Ser/Thr phosphorylation have been identified on IRS-1, and cross-talk among Tyr and Ser/Thr phosphorylations at specific residues is evidence of dynamic and complex posttranslational regulation (8, 9). Inappropriate phosphorylation of IRS-1 resulting in the disruption of interactions of IRS-1 with binding partners is implicated in the development of insulin resistance (10) and altered IGF-1 signaling in breast cancer tissue (11, 12).In addition to phosphorylation, Ser/Thr residues in IRS-1 are also dynamically modified by GlcNAc in a nutrient-responsive manner. As opposed to a negatively charged phosphate group, O-GlcNAcylation imparts a bulky, hydrophilic, electrostatically neutral moiety to Ser/Thr residues. The enzymes responsible for the incorporation and removal of the monosaccharide from proteins, O-GlcNAc-transferase and O-GlcNAcase, respectively, are localized in the cytoplasm and the nucleus of all eukaryotic cells (13, 14). Recent studies suggest that the activity of O-GlcNAc-transferase is regulated by insulin (15) and that localization of O-GlcNAc-transferase to the membrane is driven by direct association with phosphatidylinositide 3-phosphate (16). The abundance of O-GlcNAc modification on many proteins in the insulin signaling pathway increases with sustained high glucose and chronic insulin stimulation, and elevated O-GlcNAc modification of IRS-1 correlates with the development of insulin resistance in multiple cell types including 3T3-L1 adipocytes (17, 18), MIN6 pancreatic beta cells (19), Fao rat hepatoma cells (16), human aortic endothelial cells (20), and skeletal muscle (21). The impact of O-GlcNAcylation on insulin signaling and diabetic complications was reviewed recently (22, 23). The direct effect of O-GlcNAc modification on signaling via IRS-1 is not known because conditions that mimic those in the uncontrolled diabetic patient may also result in phosphorylation of IRS-1 at inhibitory sites (16, 24) and O-GlcNAc modification of other proteins in the insulin signaling pathway, such as the insulin receptor, Akt (18), FoxO (25), AMP-activated protein kinase (26), and β-catenin (17).To elucidate site-specific effects of O-GlcNAc modification on IRS-1-mediated signal transduction, we identified the sites of O-GlcNAc modification of rat and human IRS-1 by tandem mass spectrometry. To facilitate detection of the O-GlcNAc-modified peptides and assign the sites of modification, CID coupled with neutral loss-triggered MS3 and electron transfer dissociation (ETD) (27) tandem spectrometric approaches were used. Fragmentation of O-GlcNAc-modified peptides by ETD did not destroy the labile O-linkage (28) permitting direct detection of these peptides by the database searching algorithm ProteinProspector2 (29). O-GlcNAc modification occurred in close proximity to multiple SH2 domain binding motifs and within a region of IRS-1 shown previously to interact with the insulin and IGF-1 receptors (30).     

The Mycobacterium tuberculosis Ser/Thr Kinase Substrate Rv2175c Is a DNA-binding Protein Regulated by Phosphorylation

Recent efforts have underlined the role of serine/threonine protein kinases in growth, pathogenesis, and cell wall metabolism in Mycobacterium tuberculosis. Although most kinases have been investigated for their physiological roles, little information is available regarding how serine/threonine protein kinase-dependent phosphorylation regulates the activity of kinase substrates. Herein, we focused on M. tuberculosis Rv2175c, a protein of unknown function, conserved in actinomycetes, and recently identified as a substrate of the PknL kinase. We solved the solution structure of Rv2175c by multidimensional NMR and demonstrated that it possesses an original winged helix-turn-helix motif, indicative of a DNA-binding protein. The DNA-binding activity of Rv2175c was subsequently confirmed by fluorescence anisotropy, as well as in electrophoretic mobility shift assays. Mass spectrometry analyses using a combination of MALDI-TOF and LC-ESI/MS/MS identified Thr⁹ as the unique phosphoacceptor. This was further supported by complete loss of PknL-dependent phosphorylation of an Rv2175c_T9A mutant. Importantly, the DNA-binding activity was completely abrogated in a Rv2175c_T9D mutant, designed to mimic constitutive phosphorylation, but not in a mutant lacking the first 13 residues. This implies that the function of the N-terminal extension is to provide a phosphoacceptor (Thr⁹), which, following phosphorylation, negatively regulates the Rv2175c DNA-binding activity. Interestingly, the N-terminal disordered extension, which bears the phosphoacceptor, was found to be restricted to members of the M. tuberculosis complex, thus suggesting the existence of an original mechanism that appears to be unique to the M. tuberculosis complex.In response to its environment, Mycobacterium tuberculosis (M. tb)³ activates or represses the expression of a number of genes to promptly adjust to new conditions. More precisely, during the infection process, cross-talk of signals between the host and the bacterium take place, resulting in reprogramming the host signaling network. Many of these stimuli are transduced in the bacteria via sensor kinases, enabling the pathogen to adapt its cellular response to survive in hostile environments. Although the two-component systems represent the classic prokaryotic mechanism for detection and response to environmental changes, the serine/threonine and tyrosine protein kinases (STPKs) associated with their phosphatases have emerged as important regulatory systems in prokaryotic cells (1–3). M. tb contains eleven STPKs (4, 5), and most are being investigated for their physiological roles and potential application for future drug development to combat tuberculosis (6). Through phosphorylation these STPKs are also thought to play important functions in cell signaling responses as well as in essential metabolic pathways. The cell wall of M. tb plays a critical role in the defense of this pathogen in the host, and changes in cell wall composition in response to various environmental stimuli are critical to M. tb adaptation during infection. Although little is known regarding the cell wall regulatory mechanisms in M. tb, there is now an increasing body of evidence indicating that these processes largely rely on STPK-dependent mechanisms (7–9).Moreover, little information on the range of functions regulated by the STPKs is available, and the complicated mycobacterial phosphoproteome is still far from being deciphered. Understanding mycobacterial kinase biology has been severely impeded by the difficulty to identify direct kinase substrates and the subsequent characterization of the phosphorylation site(s). However, several recent studies have reported the identification and characterization of the phosphorylation sites in substrates related to various metabolic pathways in mycobacteria. These include the Fork Head associated-containing protein GarA, a key regulator of the tricarboxylic cycle (10, 11); PbpA, a penicillin-binding protein required for cell division (12); Wag31, a homologue of the cell division protein DivIVA that regulates growth, morphology, and polar cell wall biosynthesis in mycobacteria (13); the β-ketoacyl acyl carrier protein synthase mtFabH, which participates in mycolic acid biosynthesis (9); the anti-anti-sigma factor Rv0516c (14); the alternate sigma factor SigH, which is a central regulator of the response to oxidative stress (15); as well as the essential mycobacterial chaperone GroEL1 (16).Therefore, a further characterization of STPKs substrates is critical to unraveling the mechanisms by which STPK-dependent phosphorylation induces modifications, thus regulating their activity, ultimately conditioning biological responses in mycobacteria. Such studies may also provide the key to designing new inhibitors that target signal transduction pathways specific to M. tb.We recently characterized a novel substrate/kinase pair in M. tb, PknL/Rv2175c (17). pknL is associated with the ∼30-kb dcw (division cell wall) gene cluster, which encompasses several genes involved in cell wall synthesis and cell division (17, 18), raising the possibility that PknL might participate in the regulation of this gene cluster. Moreover, pknL (Rv2176) is adjacent to the Rv2175c gene, encoding a 16-kDa protein of unknown function. We further demonstrated that phosphorylation of the activation loop Thr-173 residue was required for optimal PknL-mediated phosphorylation of Rv2175c. Moreover, Rv2175c belongs to a mycobacterial “core” of 219 genes, identified by macroarray and bioinformatic analysis, common to M. tb- and Mycobacterium leprae-encoding proteins showing no similarity with proteins from other organisms. The presence of Rv2175c as a member of this set of genes emphasizes the importance of Rv2175c in the physiology of M. tb. In this context, we reasoned that the structural determination of Rv2175c would provide a valuable basis for a better understanding of the function of this protein.Therefore, we have undertaken the structural determination of Rv2175 using multidimensional NMR techniques. Herein, we provide strong evidence that Rv2175c is a DNA-binding protein and investigated how phosphorylation of a unique Thr residue in the N-terminal domain of the protein affects its DNA-binding activity.     

The Phosphoproteome of Bloodstream Form Trypanosoma brucei, Causative Agent of African Sleeping Sickness

The protozoan parasite Trypanosoma brucei is the causative agent of human African sleeping sickness and related animal diseases, and it has over 170 predicted protein kinases. Protein phosphorylation is a key regulatory mechanism for cellular function that, thus far, has been studied in T.brucei principally through putative kinase mRNA knockdown and observation of the resulting phenotype. However, despite the relatively large kinome of this organism and the demonstrated essentiality of several T. brucei kinases, very few specific phosphorylation sites have been determined in this organism. Using a gel-free, phosphopeptide enrichment-based proteomics approach we performed the first large scale phosphorylation site analyses for T.brucei. Serine, threonine, and tyrosine phosphorylation sites were determined for a cytosolic protein fraction of the bloodstream form of the parasite, resulting in the identification of 491 phosphoproteins based on the identification of 852 unique phosphopeptides and 1204 phosphorylation sites. The phosphoproteins detected in this study are predicted from their genome annotations to participate in a wide variety of biological processes, including signal transduction, processing of DNA and RNA, protein synthesis, and degradation and to a minor extent in metabolic pathways. The analysis of phosphopeptides and phosphorylation sites was facilitated by in-house developed software, and this automated approach was validated by manual annotation of spectra of the kinase subset of proteins. Analysis of the cytosolic bloodstream form T. brucei kinome revealed the presence of 44 phosphorylated protein kinases in our data set that could be classified into the major eukaryotic protein kinase groups by applying a multilevel hidden Markov model library of the kinase catalytic domain. Identification of the kinase phosphorylation sites showed conserved phosphorylation sequence motifs in several kinase activation segments, supporting the view that phosphorylation-based signaling is a general and fundamental regulatory process that extends to this highly divergent lower eukaryote.Phosphorylation by eukaryotic protein kinases (PKs)¹ is the most intensively studied posttranslational modification in eukaryotic cells mainly because reversible phosphorylation regulates almost every cellular process (1). PKs make up one of the largest protein superfamilies, comprising the protein-serine/threonine kinases and protein-tyrosine kinases (2).Knowledge of the fundamental role of protein kinases for cell survival in multicellular organisms has led to functional studies of this enzyme class in parasitic protozoa to search for alternative drug targets to treat tropical diseases (3). Trypanosoma brucei causes human African trypanosomiasis (also known as African sleeping sickness) and is responsible for ∼30,000 deaths per annum (4). Initial bioinformatics analysis of the T. brucei genome revealed the presence of 156 conventional protein kinases (ePks) and 20 atypical protein kinases (aPKs) (5). Our characterization of the T. brucei genome using a multilevel hidden Markov model (HMM) library of the kinase catalytic domain (6) followed by manual curation identified 170 ePKs and 12 aPKs.Over the past few years, research on T. brucei protein kinases has largely focused on cell cycle-regulating enzymes because of their potential as targets for African sleeping sickness (3, 7). Indeed several protein kinases have been shown to be essential for the organism (8–12). Most of the above mentioned protein kinases were characterized using techniques such as RNA interference, overexpression of the kinase and/or a kinase-dead version, in vitro and in vivo kinase assays, and immunofluorescence microscopy. However, despite these encouraging investigations, the literature for T. brucei proteins with a predicted role in signal transduction lacks information about their in vivo phosphorylation sites. Knowledge of protein phosphorylation sites, however, is crucial for a complete functional characterization of protein kinases and kinase-substrate relationships (1, 13).At the start of this study, only seven phosphorylation sites had been mapped in any of the trypanosomatids: Mehlert et al. (14) had found that six of seven threonine residues of the Gly-Pro-Glu-Glu-Thr repeat-procyclin are phosphorylated using MALDI mass spectrometry, and da Cunha et al. (15) had determined one phosphorylation site at Ser¹² of Trypanosoma cruzi histone H1 protein by electrospray MS. Over the past few years phosphoproteome studies in other organisms using phosphopeptide enrichment in combination with high accuracy mass spectrometry have revealed thousands of novel protein phosphorylation sites and given new insights into the extent and function of this posttranslational modification (16–20).In this study, we used a gel-free peptide-based phosphoproteomics approach to identify 491 trypanosomal phosphoproteins and, where possible, their precise phosphorylation sites. In addition to phosphorylation on serine and threonine residues, we identified several phosphorylated tyrosine residues in T. brucei proteins despite the lack of conventional tyrosine kinases in the T. brucei genome (5). Furthermore phosphorylation in the activation segment of protein kinases was found to be conserved in T. brucei, suggesting that at least some signaling pathways described in other eukaryotes are conserved in this ancient and highly divergent eukaryote.     

Phosphorylation-induced Conformational Changes in Rap1b: ALLOSTERIC EFFECTS ON SWITCH DOMAINS AND EFFECTOR LOOP*

Rap1b has been implicated in the transduction of the cAMP mitogenic response. Agonists that increase intracellular cAMP rapidly activate (i.e. GTP binding) and phosphorylate Rap1b on Ser¹⁷⁹ at its C terminus. cAMP-dependent protein kinase (PKA)-mediated phosphorylation of Rap1b is required for cAMP-dependent mitogenesis, tumorigenesis, and inhibition of AKT activity. However, the role of phosphorylation still remains unknown. In this study, we utilized amide hydrogen/deuterium exchange mass spectroscopy (DXMS) to assess potential conformational changes and/or mobility induced by phosphorylation. We report here DXMS data comparing exchange rates for PKA-phosphorylated (Rap1-P) and S179D phosphomimetic (Rap1-D) Rap1b proteins. Rap1-P and Rap1-D behaved exactly the same, revealing an increased exchange rate in discrete regions along the protein; these regions include a domain around the phosphorylation site and unexpectedly the two switch loops. Thus, local effects induced by Ser¹⁷⁹ phosphorylation communicate allosterically with distal domains involved in effector interaction. These results provide a mechanistic explanation for the differential effects of Rap1 phosphorylation by PKA on effector protein interaction.Rap1b, a member of the Ras superfamily of small G proteins, is a GTPase that acts as a molecular on/off switch for the transduction of several external stimuli by alternating from an inactive GDP-bound to an active GTP-bound state (1, 2). Rap1 activation is mediated by several second messengers, growth factors, cytokines, and cell adhesion molecules. The steady-state level of Rap1-GTP is tightly regulated by a family of guanine nucleotide exchange factors that catalyze the otherwise slow dissociation of GDP (i.e. activation) and GTPase-activating proteins, which stimulate the rather slow intrinsic GTPase catalytic activity (i.e. inactivation) (3). GTP binding is coupled to conformational changes in two well defined regions, the switch I (residues 30–40) and switch II (residues 60–76) domains, responsible for high affinity interaction with effector molecules (4, 5) and thus downstream signal transduction.cAMP is one among several pathways leading to Rap1 activation (6). cAMP exerts both mitogenic and anti-mitogenic responses in different cell types, and Rap1 activation is required downstream of cAMP in both scenarios (7, 8). Elevation of intracellular cAMP levels activates cAMP-dependent protein kinase (PKA)⁴ and Epac (exchange protein activated by cAMP), a Rap guanine nucleotide exchange factor (9). Expression of Rap1b in cells where cAMP is mitogenic is associated with an increase in cAMP-mediated G₁/S phase entry (7, 10), and both biochemical events, Rap activation and phosphorylation at Ser¹⁷⁹, are synergistically required for this action (11).PKA substrates able to modulate Rap1 activity (i.e. Src/C3G recruitment and GTPase-activating protein) were recently reported (12, 13). However, the role of PKA-dependent Rap1 phosphorylation at Ser¹⁷⁹ is still unknown. Rap1 phosphorylation does not affect its overall intracellular localization, its basal GTP/GDP exchange reaction, its intrinsic rate of GTP hydrolysis, or its ability to be stimulated by a cytosolic Rap GTPase-activating protein (10); however, several reports suggest that Rap1 phosphorylation is able to modulate its association with some binding partners, namely cytochrome b₅₅₈ (14) and Raf1 (15). The mechanism by which a modification of Ser¹⁷⁹ at the C-terminal end of the molecule affects the regions involved with effector interaction at its N terminus is for the moment unclear.In this study, we report a global assessment of the effects of Ser¹⁷⁹ phosphorylation on conformational change/mobility analyzed by hydrogen/deuterium exchange mass spectrometry (DXMS). The results are consistent with an allosteric effect of the C terminus (containing Ser¹⁷⁹) to the switch loops/effector domain.