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).     

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.     

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.     

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.     

Dbf4-Cdc7 Phosphorylation of Mcm2 Is Required for Cell Growth

The Dbf4-Cdc7 kinase (DDK) is required for the activation of the origins of replication, and DDK phosphorylates Mcm2 in vitro. We find that budding yeast Cdc7 alone exists in solution as a weakly active multimer. Dbf4 forms a likely heterodimer with Cdc7, and this species phosphorylates Mcm2 with substantially higher specific activity. Dbf4 alone binds tightly to Mcm2, whereas Cdc7 alone binds weakly to Mcm2, suggesting that Dbf4 recruits Cdc7 to phosphorylate Mcm2. DDK phosphorylates two serine residues of Mcm2 near the N terminus of the protein, Ser-164 and Ser-170. Expression of mcm2-S170A is lethal to yeast cells that lack endogenous MCM2 (mcm2Δ); however, this lethality is rescued in cells harboring the DDK bypass mutant mcm5-bob1. We conclude that DDK phosphorylation of Mcm2 is required for cell growth.The Cdc7 protein kinase is required throughout the yeast S phase to activate origins (1, 2). The S phase cyclin-dependent kinase also activates yeast origins of replication (3–5). It has been proposed that Dbf4 activates Cdc7 kinase in S phase, and that Dbf4 interaction with Cdc7 is essential for Cdc7 kinase activity (6). However, it is not known how Dbf4-Cdc7 (DDK)² acts during S phase to trigger the initiation of DNA replication. DDK has homologs in other eukaryotic species, and the role of Cdc7 in activation of replication origins during S phase may be conserved (7–10).The Mcm2-7 complex functions with Cdc45 and GINS to unwind DNA at a replication fork (11–15). A mutation of MCM5 (mcm5-bob1) bypasses the cellular requirements for DBF4 and CDC7 (16), suggesting a critical physiologic interaction between Dbf4-Cdc7 and Mcm proteins. DDK phosphorylates Mcm2 in vitro with proteins purified from budding yeast (17, 18) or human cells (19). Furthermore, there are mutants of MCM2 that show synthetic lethality with DBF4 mutants (6, 17), suggesting a biologically relevant interaction between DBF4 and MCM2. Nevertheless, the physiologic role of DDK phosphorylation of Mcm2 is a matter of dispute. In human cells, replacement of MCM2 DDK-phosphoacceptor residues with alanines inhibits DNA replication, suggesting that Dbf4-Cdc7 phosphorylation of Mcm2 in humans is important for DNA replication (20). In contrast, mutation of putative DDK phosphorylation sites at the N terminus of Schizosaccharomyces pombe Mcm2 results in viable cells, suggesting that phosphorylation of S. pombe Mcm2 by DDK is not critical for cell growth (10).In budding yeast, Cdc7 is present at high levels in G₁ and S phase, whereas Dbf4 levels peak in S phase (18, 21, 22). Furthermore, budding yeast DDK binds to chromatin during S phase (6), and it has been shown that Dbf4 is required for Cdc7 binding to chromatin in budding yeast (23, 24), fission yeast (25), and Xenopus (9). Human and fission yeast Cdc7 are inert on their own (7, 8), but Dbf4-Cdc7 is active in phosphorylating Mcm proteins in budding yeast (6, 26), fission yeast (7), and human (8, 10). Based on these data, it has been proposed that Dbf4 activates Cdc7 kinase in S phase and that Dbf4 interaction with Cdc7 is essential for Cdc7 kinase activity (6, 9, 18, 21–24). However, a mechanistic analysis of how Dbf4 activates Cdc7 has not yet been accomplished. For example, the multimeric state of the active Dbf4-Cdc7 complex is currently disputed. A heterodimer of fission yeast Cdc7 (Hsk1) in complex with fission yeast Dbf4 (Dfp1) can phosphorylate Mcm2 (7). However, in budding yeast, oligomers of Cdc7 exist in the cell (27), and Dbf4-Cdc7 exists as oligomers of 180 and 300 kDa (27).DDK phosphorylates the N termini of human Mcm2 (19, 20, 28), human Mcm4 (10), budding yeast Mcm4 (26), and fission yeast Mcm6 (10). Although the sequences of the Mcm N termini are poorly conserved, the DDK sites identified in each study have neighboring acidic residues. The residues of budding yeast Mcm2 that are phosphorylated by DDK have not yet been identified.In this study, we find that budding yeast Cdc7 is weakly active as a multimer in phosphorylating Mcm2. However, a low molecular weight form of Dbf4-Cdc7, likely a heterodimer, has a higher specific activity for phosphorylation of Mcm2. Dbf4 or DDK, but not Cdc7, binds tightly to Mcm2, suggesting that Dbf4 recruits Cdc7 to Mcm2. DDK phosphorylates two serine residues of Mcm2, Ser-164 and Ser-170, in an acidic region of the protein. Mutation of Ser-170 is lethal to yeast cells, but this phenotype is rescued by the DDK bypass mutant mcm5-bob1. We conclude that DDK phosphorylation of Ser-170 of Mcm2 is required for budding yeast growth.     

Effect of Pin1 or Microtubule Binding on Dephosphorylation of FTDP-17 Mutant Tau

Neurodegenerative tauopathies, including Alzheimer disease, are characterized by abnormal hyperphosphorylation of the microtubule-associated protein Tau. One group of tauopathies, known as frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), is directly associated with mutations of the gene tau. However, it is unknown why mutant Tau is highly phosphorylated in the patient brain. In contrast to in vivo high phosphorylation, FTDP-17 Tau is phosphorylated less than wild-type Tau in vitro. Because phosphorylation is a balance between kinase and phosphatase activities, we investigated dephosphorylation of mutant Tau proteins, P301L and R406W. Tau phosphorylated by Cdk5-p25 was dephosphorylated by protein phosphatases in rat brain extracts. Compared with wild-type Tau, R406W was dephosphorylated faster and P301L slower. The two-dimensional phosphopeptide map analysis suggested that faster dephosphorylation of R406W was due to a lack of phosphorylation at Ser-404, which is relatively resistant to dephosphorylation. We studied the effect of the peptidyl-prolyl isomerase Pin1 or microtubule binding on dephosphorylation of wild-type Tau, P301L, and R406W in vitro. Pin1 catalyzes the cis/trans isomerization of phospho-Ser/Thr-Pro sequences in a subset of proteins. Dephosphorylation of wild-type Tau was reduced in brain extracts of Pin1-knockout mice, and this reduction was not observed with P301L and R406W. On the other hand, binding to microtubules almost abolished dephosphorylation of wild-type and mutant Tau proteins. These results demonstrate that mutation of Tau and its association with microtubules may change the conformation of Tau, thereby suppressing dephosphorylation and potentially contributing to the etiology of tauopathies.One of hallmarks of Alzheimer disease (AD)³ pathology is neurofibrillary tangles, which are composed of paired helical filaments (PHFs), aggregates of the abnormally phosphorylated microtubule-associated protein Tau. Intracellular inclusions comprising Tau are also found in several other neurodegenerative diseases, including Pick disease, progressive supranuclear palsy, corticobasal degeneration, and frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), collectively called tauopathies (1–3). Identification of Tau as a causative gene of the inherited tauopathy FTDP-17 reveals that Tau mutation is sufficient to cause disease (4–6). However, the impact Tau mutations have on neurodegeneration remains unknown.Tau proteins in inclusions are hyperphosphorylated, and extensive studies have identified the phosphorylation sites; for example, more than 20 sites have been identified in PHF-Tau obtained from AD brains (7, 8). Tau can be phosphorylated by a variety of protein kinases, including glycogen synthase kinase 3β (GSK3β), cyclin-dependent kinase 5 (Cdk5), mitogen-activated protein kinase, cAMP-dependent protein kinase (PKA), microtubule affinity regulating kinase, and others (9–11). Tau is predominantly phosphorylated on the Ser or Thr residue in Ser/Thr-Pro sequences, suggesting the involvement of proline-directed protein kinases such as GSK3β and Cdk5 in hyperphosphorylation. A critical question is how mutations in Tau induce hyperphosphorylation in brain (12). Early phosphorylation experiments in vitro and in cultured cells have shown that mutant Tau is less phosphorylated than wild-type (WT) Tau (13–18). However, two later studies demonstrated higher phosphorylation of mutant Tau using brain extracts as a source of protein kinases in the presence of protein phosphatase inhibitor okadaic acid (19) or in immortalized cortical cells (20). However, it is not fully understood how mutant Tau becomes highly phosphorylated in vivo.Tau hyperphosphorylation could also be attributed to reduced dephosphorylation activity. Tau is dephosphorylated in vitro by any of the major four classes of protein phosphatases, PP1, PP2A, PP2B, and PP2C, but PP2A is thought to be the major protein phosphatase that regulates Tau phosphorylation state in brains (21–23). PP2A activity reportedly is decreased in AD brain (24–26), and highly phosphorylated Tau in PHF is relatively resistant to dephosphorylation by PP2A (27). Few studies have been done on dephosphorylation of mutant Tau, however, and thus the mechanism remains unclear. One putative factor involved in mutant Tau dephosphorylation is the peptidyl-prolyl isomerase Pin1. Pin1 catalyzes the cis/trans isomerization of phospho-Ser/Thr-Pro sequences in a subset of proteins (28, 29). Pin1 is involved in AD pathogenesis as shown by the fact that it is found in neurofibrillary tangles and that Tau is hyperphosphorylated in Pin1-deficient mouse brains (30). Pin1 is indicated to facilitate Tau dephosphorylation via PP2A by binding to the phospho-Thr-231-Pro or phospho-Thr-212-Pro site (31–33). The effect of Pin1 on the stability of mutant Tau was recently reported (34), but a detailed analysis of Pin1 action on mutant Tau has not been reported. Another possible factor affecting dephosphorylation of mutant Tau is the binding to microtubules. We previously showed that phosphorylation of Tau is stimulated upon binding to microtubules (35). We thus hypothesized that binding to microtubules may also affect the extent of Tau dephosphorylation.Here, we examined the effects of Pin1 and binding to microtubules on dephosphorylation of WT and FTDP-17 mutant (P301L and R406W) Tau proteins that had been phosphorylated by Cdk5-p25 or Cdk5-p35. P301L and R406W are two distinct types of FTDP-17 mutants that have been studied well. We show for the first time how the regulation of Tau dephosphorylation can contribute to the observed Tau hyperphosphorylation in tauopathies.     

Phosphorylation Dependence of Hsp27 Multimeric Size and Molecular Chaperone Function

The molecular chaperone Hsp27 exists as a distribution of large oligomers that are disassembled by phosphorylation at Ser-15, -78, and -82. It is controversial whether the unphosphorylated Hsp27 or the widely used triple Ser-to-Asp phospho-mimic mutant is the more active molecular chaperone in vitro. This question was investigated here by correlating chaperone activity, as measured by the aggregation of reduced insulin or α-lactalbumin, with Hsp27 self-association as monitored by analytical ultracentrifugation. Furthermore, because the phospho-mimic is generally assumed to reproduce the phosphorylated molecule, the size and chaperone activity of phosphorylated Hsp27 were compared with that of the phospho-mimic. Hsp27 was triply phosphorylated by MAPKAP-2 kinase, and phosphorylation was tracked by urea-PAGE. An increasing degree of suppression of insulin or α-lactalbumin aggregation correlated with a decreasing Hsp27 self-association, which was the least for phosphorylated Hsp27 followed by the mimic followed by the unphosphorylated protein. It was also found that Hsp27 added to pre-aggregated insulin did not reverse aggregation but did inhibit these aggregates from assembling into even larger aggregates. This chaperone activity appears to be independent of Hsp27 phosphorylation. In conclusion, the most active chaperone of insulin and α-lactalbumin was the Hsp27 (elongated) dimer, the smallest Hsp27 subunit observed under physiological conditions. Next, the Hsp27 phospho-mimic is only a partial mimic of phosphorylated Hsp27, both in self-association and in chaperone function. Finally, the efficient inhibition of insulin aggregation by Hsp27 dimer led to the proposal of two models for this chaperone activity.Oligomeric heat shock protein 27 (Hsp27)² is a ubiquitous mammalian protein with a variety of functions in health and disease (1–8). These functions include ATP-independent chaperone activity in response to environmental stress, e.g. heat shock and oxidative stress, control of apoptosis, and regulation of actin cytoskeleton dynamics. Hsp27 is a member of the α-crystallin small heat shock protein family of which αB-crystallin is the archetype. These proteins are characterized by an α-crystallin domain of 80–90 residues consisting of roughly eight β-strands that form an intermolecular β-sheet interaction interface within a dimer, the basic building subunit of the oligomer (2, 4, 9–11).Hsp27 is in equilibrium between high molecular weight oligomers and much lower molecular weight multimers. It has been reported that unphosphorylated Hsp27 includes predominantly a distribution of high molecular species ranging in size from 12-mer to 35-mer (12–19). Phosphorylation of Hsp27 at serines 15, 78, and 82 by the p38-activated MAPKAP-2 kinase (20–22) or the use of the triple Ser-to-Asp phospho-mimic results in a major shift in the equilibrium toward much smaller multimers (23) and in an alteration of its function (1, 3, 6, 7, 24, 25). The size distribution of the smaller species has been reported to be between monomer and tetramer (12–16, 18, 19).Small heat shock proteins, including Hsp27, behave as ATP-independent molecular chaperones during cellular heat shock. They bind partially unfolded proteins and prevent their aggregation until the proteins can be refolded by larger ATP-dependent chaperones or are digested (7, 8, 26). This function includes the up-regulation and/or phosphorylation of Hsp27.It is not entirely clear what the role of Hsp27 size and phosphorylation state plays in its heat shock function because there are conflicting results in the literature. Some in vitro studies concluded that the unphosphorylated oligomeric Hsp27 (or the murine isoform Hsp25) protects proteins against aggregation better than does the phosphorylation mimic (13, 19, 27), whereas others found no difference (16, 28, 29), and still other studies found that the mimic protects better than does the unphosphorylated wild type (27, 30, 31). In-cell studies found that phosphorylation of Hsp27 was essential for thermo-protection of actin filaments (32), and the Hsp27 phosphorylation mimic decreased inclusion body formation better than did unphosphorylated Hsp27 (33). This study was undertaken to investigate the molecular chaperone function of Hsp27 by correlating chaperone activity with Hsp27 size and by comparing fully phosphorylated Hsp27 with its phospho-mimic.     

Comprehensive Analysis of Phosphorylation Sites in Tensin1 Reveals Regulation by p38MAPK

Tensin1 is the archetype of a family of focal adhesion proteins. Tensin1 has a phosphotyrosine binding domain that binds the cytoplasmic tail of β-integrin, a Src homology 2 domain that binds focal adhesion kinase, p130Cas, and the RhoGAP called deleted in liver cancer-1, a phosphatase and tensin homology domain that binds protein phosphatase-1α and other regions that bind F-actin. The association between tensin1 and these partners affects cell polarization, migration, and invasion. In this study we analyzed the phosphorylation of human S-tag-tensin1 expressed in HEK293 cells by mass spectrometry. Peptides covering >90% of the sequence initially revealed 50 phosphorylated serine/phosphorylated threonine (pSer/pThr) but no phosphorylated tyrosine (pTyr) sites. Addition of peroxyvanadate to cells to inhibit protein tyrosine phosphatases exposed 10 pTyr sites and addition of calyculin A to cells to inhibit protein phosphatases type 1 and 2A gave a total of 62 pSer/pThr sites. We also characterized two sites modified by O-linked N-acetylglucosamine. Tensin1 F302A, which does not bind protein phosphatase-1, showed > twofold enhanced phosphorylation of seven sites. The majority of pSer/pThr have adjacent proline (Pro) residues and we show endogenous p38 mitogen activated protein kinase (MAPK) associated with and phosphorylated tensin1 in an in vitro kinase assay. Recombinant p38α MAPK also phosphorylated S-tag-tensin1, resulting in decreased binding with deleted in liver cancer-1. Activation of p38 MAPK in cells by sorbitol-induced hyperosmotic stress increased phosphorylation of S-tag-tensin1, which reduced binding to deleted in liver cancer-1 and increased binding to endogenous pTyr proteins, including p130Cas and focal adhesion kinase. These data demonstrate that tensin1 is extensively phosphorylated on Ser/Thr residues in cells and phosphorylation by p38 MAPK regulates the specificity of the tensin1 Src homology 2 domain for binding to different proteins. Tensin1 provides a hub for connecting signaling pathways involving p38 MAP kinase, tyrosine kinases and RhoGTPases.Tensin1 is a protein localized at focal adhesions that acts as a scaffold for signaling (1). The tensin1 phosphotyrosine binding (PTB)1 domain binds the cytoplasmic tail of β-integrin (2), presumed to be the basis for focal adhesion localization. Human tensin1 interacts with actin by capping the barbed ends and cross-linking actin filaments through two different actin binding regions (3). Actin binding regions were identified in chicken tensin1 at residues 1–263, 263–463, and 889–1143 (4). The C terminus region of tensin1, as well as family members tensin2, tensin3, and c-ten, has adjacent Src homology 2 (SH2) and PTB domains that interact with the tyrosine phosphorylated proteins Dok2 and PDK1 (5) as well as PI3 kinase, p130Cas, and focal adhesion kinase (FAK) (6), thereby posing a role for tensin1 in multiple signal transduction pathways. The N-terminal region of tensin1 contains a domain that is related in sequence to the tumor suppressor protein and PIP3 phosphatase called phosphatase and tensin homologue (PTEN) (3). This domain of tensin1 binds the alpha isoform of protein phosphatase 1 (PP1) (7), the major protein Ser/Thr phosphatase in cells that regulates a variety of signaling pathways. The SH2 domain of tensin1 also associates with a RhoGAP protein called deleted in liver cancer-1 (DLC-1) but does not require Tyr phosphorylation of DLC-1 (8). DLC-1 has a role in cell migration and is a negative regulator of tumor formation (8–10). Human breast carcinoma, prostate carcinoma, head and neck squamous cell carcinoma, and melanoma all exhibit reduced expression of tensin1, suggesting a tumor suppressor action (11). In addition, various cancer cell lines do not express detectable levels of tensin1 protein relative to normal fibroblasts that have abundant expression (1, 7). Re-expression of tensin1 in cancer cells promoted formation of focal adhesions (4) and decreased migration and invasion of MDA MB 231 human breast cancer cells (12). Taken together, these studies support a model for tensin1 as a tumor suppressor that acts as a scaffold protein for various signaling enzymes.Tensin1 was first shown to be tyrosine phosphorylated following concentration by immunoprecipitation and immunoblotting with a pTyr antibody (6). Tyrosine phosphorylation of tensin1 was only detected if fibroblasts were plated on fibronectin, laminin, or vitronectin (13), suggesting that tensin1 tyrosine phosphorylation depends on integrin-mediated signaling. Jiang et al. (14) showed increased tyrosine phosphorylation of tensin1 when cells were treated with platelet-derived growth factor. In addition, epidermal growth factor treatment of human gastric epithelial cells stimulated tyrosine phosphorylation of tensin1 and this stimulation was inhibited with the nonsteroidal anti-inflammatory drug indomethacin (15). Cells transformed by the oncogene p210BCR/ABL contained tyrosine phosphorylated tensin1 (16). Treatment of rat aortic smooth muscle cells with angiotensin or thrombin also showed an increase in tensin1 tyrosine phosphorylation (17). Rapid turnover of pTyr by phosphatases presumably keeps tensin1 pTyr levels low in cells following stimulation. Different publications report tensin1 is phosphorylated on Ser and Thr residues, but data supporting these claims was not shown (1, 3, 18, 19). Phosphoproteomics implementing shotgun mass spectrometry techniques have turned up as many as 20 pTyr, 30 pSer, and 8 pThr peptides from human tensin (www.phosphosite.org). However, to date no comprehensive analysis of tensin1 phosphorylation has been reported.We previously identified residue F302 in the KVEF motif in tensin1 as necessary for PP1α binding (12). Tensin1 F302A showed a reduced electrophoretic mobility in SDS-PAGE compared with tensin1 wild type, suggesting an increase in tensin1 phosphorylation because of absence of bound PP1. We also observed less DLC-1 binding to tensin1 F302A, but it is not known whether this was because of an increase in tensin1 phosphorylation (12). The tensin1 F302A did not suppress cancer cell invasion like tensin1 wild type (12), and this could be because of loss of PP1 binding, or less DLC-1 binding, or changes in phosphorylation.In the present study we comprehensively analyze the phosphorylation of human S-tag-tensin1. Addition of phosphatase inhibitors to cells is shown to enhance phosphorylation to yield a total of 62 Ser/Thr phosphorylation sites and expose 10 Tyr sites not otherwise seen. The majority of Ser/Thr sites have adjacent proline residues and we identify p38α MAPK activity associated with tensin1. The p38MAPK phosphorylation of tensin1 alters binding of DLC-1, p130Cas and FAK. Our results demonstrate that tensin1 is extensively phosphorylated on Ser/Thr residues in addition to Tyr residues and this phosphorylation alters association with its SH2 domain binding partners.     

Identification and Functional Characterization of Protein Kinase A-catalyzed Phosphorylation of Potassium Channel Kv1.2 at Serine 449

Vascular smooth muscle Kv1 delayed rectifier K⁺ channels (K_DR) containing Kv1.2 control membrane potential and thereby regulate contractility. Vasodilatory agonists acting via protein kinase A (PKA) enhance vascule smooth muscle Kv1 activity, but the molecular basis of this regulation is uncertain. We characterized the role of a C-terminal phosphorylation site, Ser-449, in Kv1.2 expressed in HEK 293 cells by biochemical and electrophysiological methods. We found that 1) in vitro phosphorylation of Kv1.2 occurred exclusively at serine residues, 2) one major phosphopeptide that co-migrated with ⁴⁴⁹pSASTISK was generated by proteolysis of in vitro phosphorylated Kv1.2, 3) the peptide ⁴⁴⁵KKSRSASTISK exhibited stoichiometric phosphorylation by PKA in vitro, 4) matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectroscopy (MS) and MS/MS confirmed in vitro Ser-449 phosphorylation by PKA, 5) in situ phosphorylation at Ser-449 was detected in HEK 293 cells by MALDI-TOF MS followed by MS/MS. MIDAS (multiple reaction monitoring-initiated detection and sequencing) analysis revealed additional phosphorylated residues, Ser-440 and Ser-441, 6) in vitro ³²P incorporation was significantly reduced in Kv1.2-S449A, Kv1.2-S449D, and Kv1.2-S440A/S441A/S449A mutant channels, but Kv1.2-S440A/S441A was identical to wild-type Kv1.2 (Kv1.2-WT), and 7) bath applied 8-Br-cAMP or dialysis with PKA catalytic subunit (cPKA) increased Kv1.2-WT but not Kv1.2-S449A current amplitude. cPKA increased Kv1.2-WT current in inside-out patches. Rp-CPT-cAMPS reduced Kv1.2-WT current, blocked the increase due to 8-Br-cAMP, but had no effect on Kv1.2-S449A. cPKA increased current due to double mutant Kv1.2-S440A/S441A but had no effect on Kv1.2-S449D or Kv1.2-S440A/S441A/S449A. We conclude that Ser-449 in Kv1.2 is a site of PKA phosphorylation and a potential molecular mechanism for Kv1-containing K_DR channel modulation by agonists via PKA activation.Voltage-gated K⁺ channels (Kv)⁴ composed of members of the Kv1 family (Kv1) are expressed in vascular smooth muscle (VSM) and other excitable cells, where they play an important role in controlling membrane potential, a key mechanism for regulation of contraction and arterial diameter (1). Steady-state Ca²⁺ influx through voltage-gated Ca²⁺ channels, resulting in elevated cytosolic Ca²⁺ concentration and contraction, requires sustained depolarization. In contrast, Kv1 channel activation induces hyperpolarization, reduces voltage-gated Ca²⁺ channel open probability, and promotes relaxation (1–3).Phosphorylation of ion channels by protein kinases is an important mechanism by which ion channel gating (opening and closing) and, thereby, membrane potential are modulated by cellular signaling pathways. VSM Kv1 channels containing Kv1.2-Kv1.5 (4–6) alone or with Kv1.6 (7) contribute to control of arterial diameter by vasoconstrictor and vasodilator stimuli (8–13). For example, hyperpolarization and relaxation induced by elevations in intracellular (cAMP) are suppressed by the block of Kv1 channels (14–18), and the activity of Kv1-containing VSM K_DR is enhanced by protein kinase A (PKA) (10–13, 19–22). However, the molecular basis of this regulation is unclear.Previous studies showed that gating of Kv1.2 channels expressed in Xenopus oocytes is stimulated by PKA (23), similar to VSM K_DR channels, which contain this pore-forming Kv1 subunit (7–13). There are six potential PKA phosphorylation sites within the cytoplasmic regions of Kv1.2 (see Fig. 1, A and B) based on the consensus motif Arg/Lys-Xaa-Xaa-Ser/Thr (24). An N-terminal residue, Thr-46, was proposed to be the site of PKA phosphorylation, as mutation to valine (T46V) prevented modulation by β-adrenoreceptor activation or exposure to the catalytic subunit of PKA (cPKA) (23). However, this conclusion has been questioned based on structural analysis of the N-terminal domain and the positive shift in voltage-dependent gating produced by the T46V mutation (25). Sites Ser-440, Ser-441, and Ser-449 near the C terminus were recently identified to be phosphorylated in Kv1.2 immunoprecipitates derived from brain as well as transfected human embryonic kidney (HEK 293) and COS-1 cells using a proteomic approach (26). Phosphorylation of these residues was suggested to be important for trafficking to the cell membrane (26), but the kinase(s) involved was not identified. cAMP/PKA-dependent signaling was reported to alter surface expression of Kv1.2, but phosphorylation at Ser-440 and Ser-449 was not required (27).Open in a separate windowFIGURE 1.Identification of potential PKA consensus phosphorylation sites in Kv1.2 and immunoprecipitation of Myc-tagged wild-type and mutant Kv1.2. A, schematic of Kv1.2 with candidate PKA phosphorylation sites indicated by asterisks, and Ser/Thr residues relevant to the study are labeled. B, amino acid sequence of Kv1.2 indicating transmembrane domains (boxes), cytoplasmic domains (italics) assessed by MIDAS for phosphorylated residues, candidate PKA consensus sites (bold), and potential phosphorylated Ser/Thr residues (underlined). C, immunoblot (IB) analysis of untagged Kv1.2-WT and Myc-tagged WT and mutant constructs using anti-Kv1.2. D, immunoblot identification of untagged Kv1.2-WT and Myc-tagged WT and mutant constructs by anti-Kv1.2 in anti-Myc immunoprecipitates (IP).In the present study we used a combination of biochemical, mutagenesis, and electrophysiological analyses to 1) determine whether Kv1.2 is a substrate for PKA, 2) identify the PKA phosphorylation site(s) involved, and 3) determine whether phosphorylation of the identified residue(s) mediates PKA-dependent changes in Kv1.2 current amplitude.     

Use of Quantitative Mass Spectrometric Analysis to Elucidate the Mechanisms of Phospho-priming and Auto-activation of the Checkpoint Kinase Rad53 in Vivo

The cell cycle checkpoint kinases play central roles in the genome maintenance of eukaryotes. Activation of the yeast checkpoint kinase Rad53 involves Rad9 or Mrc1 adaptor-mediated phospho-priming by Mec1 kinase, followed by auto-activating phosphorylation within its activation loop. However, the mechanisms by which these adaptors regulate priming phosphorylation of specific sites and how this then leads to Rad53 activation remain poorly understood. Here we used quantitative mass spectrometry to delineate the stepwise phosphorylation events in the activation of endogenous Rad53 in response to S phase alkylation DNA damage, and we show that the two Rad9 and Mrc1 adaptors, the four N-terminal Mec1-target TQ sites of Rad53 (Rad53-SCD1), and Rad53-FHA2 coordinate intimately for optimal priming phosphorylation to support substantial Rad53 auto-activation. Rad9 or Mrc1 alone can mediate surprisingly similar Mec1 target site phosphorylation patterns of Rad53, including previously undetected tri- and tetraphosphorylation of Rad53-SCD1. Reducing the number of TQ motifs turns the SCD1 into a proportionally poorer Mec1 target, which then requires the presence of both Mrc1 and Rad9 for sufficient priming and auto-activation. The phosphothreonine-interacting Rad53-FHA domains, particularly FHA2, regulate phospho-priming by interacting with the checkpoint mediators but do not seem to play a major role in the phospho-SCD1-dependent auto-activation step. Finally, mutation of all four SCD1 TQ motifs greatly reduces Rad53 activation but does not eliminate it, and residual Rad53 activity in this mutant is dependent on Rad9 but not Mrc1. Altogether, our results provide a paradigm for how phosphorylation site clusters and checkpoint mediators can be involved in the regulation of signaling relay in protein kinase cascades in vivo and elucidate an SCD1-independent Rad53 auto-activation mechanism through the Rad9 pathway. The work also demonstrates the power of mass spectrometry for in-depth analyses of molecular mechanisms in cellular signaling in vivo.Eukaryotic cells are most vulnerable to exogenous DNA-damaging agents during the S phase of the cell cycle, when unprogrammed DNA lesions interfere with the tightly choreographed DNA replication process. DNA damage during this phase leads to the activation of two overlapping checkpoint pathways in Saccharomyces cerevisiae, the DNA replication checkpoint and the intra-S-phase DNA damage checkpoint (1, 2). Phospho-priming for auto-activation of the central checkpoint kinase Rad53 by the upstream kinase Mec1/Tel1 depends on Mrc1 as an adaptor in the DNA replication checkpoint pathway and Rad9 as an adaptor in the DNA damage checkpoint pathway (3–10). Rad53, a well-accepted model system for studying the function and regulation of Chk2-like kinases, contains two forkhead-associated (FHA)¹ domains (FHA1 and -2) and two SQ/TQ cluster domains (SCD1 and -2) enriched in Mec1/Tel1-target phosphorylation sites (11–13).Mrc1 normally is a replisome component that functionally couples DNA Pol ε with Cdc45 and MCM helicase during replication fork progression (14, 15). As the replication forks are stalled by replication stress, the recruited checkpoint sensor kinase Mec1 phosphorylates the SCD of Mrc1, which abolishes its N-terminal interaction with Pol ε and enables Mrc1 to recruit Rad53 and promote Rad53 phosphorylation by Mec1 as an initial step in the activation of Rad53 in the Mrc1 branch (6, 14, 16). Alanine substitution of all Mec1 target sites of Mrc1 (designated the mrc1-AQ allele) has been shown to selectively disable its checkpoint function for Rad53 activation without affecting its DNA replication functions (4). In response to DNA damage, Rad9 is able to associate with damaged chromatin via its BRCT and Tudor domains, which tether it to Ser129-phosphorylated histone H2A (γH2A) and Lys79-methylated histone H3, respectively (17, 18). Alternatively, the recruitment of Rad9 onto damaged DNA could also be facilitated by its phosphorylation by CDK1, which enables the specific interaction of Rad9 with Dpb11, allowing the formation of the ternary complex of Dpb11, Mec1, and Rad9 (19, 20). Similar to Mrc1, Mec1 activates the adaptor function of Rad9 by phosphorylation of its SCD, which then binds to the Rad53-FHA domains to promote Rad53 phosphorylation by Mec1 (3, 5, 10).Beyond serving as scaffolds to recruit Rad53, Mrc1 and Rad9 have been shown to promote Rad53 phosphorylation by Mec1 in a dose-dependent manner in vitro (3, 16), underlining their adaptor role to enhance the enzyme–substrate (Mec1–Rad53) interaction. However, how they can specifically regulate the priming phosphorylation at specific sites and how this then leads to Rad53 activation remains poorly understood. Finally, hyperphosphorylated Rad9 has also been shown to catalyze the auto-phosphorylation of recombinant Rad53 (21), but it remains to be examined whether and how this occurs in vivo.The activation of SCD-FHA containing kinases such as human Chk2 and fission yeast Cds1 has been suggested to involve a two-step phosphorylation process: first, SCD phosphorylation by an ATM/ATR-like kinase leads to intermolecular binding to the FHA domain of another Chk2/Cds1 monomer, which then results in dimerization/oligomerization-dependent auto-phosphorylation within the kinase activation loop (22–26). In addition to the characteristic N-terminal SCD-FHA module of Chk2-like kinases, Rad53 contains another SCD2-FHA2 module C-terminal to its kinase domain. Similar to its orthologues, Rad53 activation has been proposed to depend on SCD1 phosphorylation (but not SCD2 phosphorylation) and partially redundant functions of the two FHA domains (9, 27–29). However, although Rad53-FHA1 can interact with SCD1 in a phospho-threonine (pT)-dependent manner in vitro (9, 28), it appears to be required for Rad53 activation only in G2/M-arrested cells (27, 29). In contrast, the FHA2 domain, which seems to be more important overall for Rad53 activation, does not appreciably bind phospho-SCD1 peptides in vitro (27, 28). Thus, the mechanisms by which Mrc1, Rad9, SCD1 phosphorylation, and FHA domains interact during checkpoint-dependent Rad53 priming and auto-activation remain to be elucidated.Quantitative mass spectrometric analysis has revolutionized the functional analysis of cellular signaling pathways, including site-specific phosphorylation events of key signaling molecules (30–33), but an important caveat is that MS studies often involve protein tags or nonphysiological expression levels that can interfere with normal protein functions. For example, the integration of a triple HA tag into the endogenous RAD53 gene locus has been shown to reduce Rad53 protein levels, resulting in significantly altered checkpoint activity (34). In this study we used quantitative MS analyses to dissect the stepwise phosphorylation events of endogenous, untagged Rad53 in response to MMS-induced alkylation DNA damage and replication stress during the S phase. Together with functional analyses, our results delineate how the two Mec1 adaptors Rad9 and Mrc1 can coordinate with the four SCD1 priming sites (T5, T8, T12, and T15) to regulate the phospho-priming of Rad53 by Mec1. In addition, an SCD1-priming independent Rad53 auto-activation mechanism and the specific roles of the FHA domains during Rad53 hyperphosphorylation are also elucidated in this work.