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

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

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.     

A Straight Path to Circular Proteins

Folding and stability are parameters that control protein behavior. The possibility of conferring additional stability on proteins has implications for their use in vivo and for their structural analysis in the laboratory. Cyclic polypeptides ranging in size from 14 to 78 amino acids occur naturally and often show enhanced resistance toward denaturation and proteolysis when compared with their linear counterparts. Native chemical ligation and intein-based methods allow production of circular derivatives of larger proteins, resulting in improved stability and refolding properties. Here we show that circular proteins can be made reversibly with excellent efficiency by means of a sortase-catalyzed cyclization reaction, requiring only minimal modification of the protein to be circularized.Sortases are bacterial enzymes that predominantly catalyze the attachment of surface proteins to the bacterial cell wall (1, 2). Other sortases polymerize pilin subunits for the construction of the covalently stabilized and covalently anchored pilus of the Gram-positive bacterium (3–5). The reaction catalyzed by sortase involves the recognition of short 5-residue sequence motifs, which are cleaved by the enzyme with the concomitant formation of an acyl enzyme intermediate between the active site cysteine of sortase and the carboxylate at the newly generated C terminus of the substrate (1, 6–8). In many bacteria, this covalent intermediate can be resolved by nucleophilic attack from the pentaglycine side chain in a peptidoglycan precursor, resulting in the formation of an amide bond between the pentaglycine side chain and the carboxylate at the cleavage site in the substrate (9, 10). In pilus construction, alternative nucleophiles such as lysine residues or diaminopimelic acid participate in the transpeptidation reaction (3, 4).When appended near the C terminus of proteins that are not natural sortase substrates, the recognition sequence of Staphylococcus aureus sortase A (LPXTG) can be used to effectuate a sortase-catalyzed transpeptidation reaction using a diverse array of artificial glycine-based nucleophiles (Fig. 1). The result is efficient installation of a diverse set of moieties, including lipids (11), carbohydrates (12), peptide nucleic acids (13), biotin (14), fluorophores (14, 15), polymers (16), solid supports (16–18), or peptides (15, 19) at the C terminus of the protein substrate. During the course of our studies to further expand sortase-based protein engineering, we were struck by the frequency and relative ease with which intramolecular transpeptidation reactions were occurring. Specifically, proteins equipped with not only the LPXTG motif but also N-terminal glycine residues yielded covalently closed circular polypeptides (Fig. 1). Similar reactivity using sortase has been described in two previous cases; however, rigorous characterization of the circular polypeptides was absent (16, 20). The circular proteins in these reports were observed as minor components of more complex reaction mixtures, and the cyclization reaction itself was not optimized.Open in a separate windowFIGURE 1.Protein substrates equipped with a sortase A recognition sequence (LPXTG) can participate in intermolecular transpeptidation with synthetic oligoglycine nucleophiles (left) or intramolecular transpeptidation if an N-terminal glycine residue is present (right).Here we describe our efforts toward applying sortase-catalyzed transpeptidation to the synthesis of circular and oligomeric proteins. This method has general applicability, as illustrated by successful intramolecular reactions with three structurally unrelated proteins. In addition to circularization of individual protein units, the multiprotein complex AAA-ATPase p97/VCP/CDC48, with six identical subunits containing the LPXTG motif and an N-terminal glycine, was found to preferentially react in daisy chain fashion to yield linear protein fusions. The reaction exploited here shows remarkable similarities to the mechanisms proposed for circularization of cyclotides, small circular proteins that have been isolated from plants (21–23).     

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.     

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.     

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.     

Evolutionary Constraints of Phosphorylation in Eukaryotes,Prokaryotes, and Mitochondria

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

Phosphoproteome Analysis Reveals Regulatory Sites in Major Pathways of Cardiac Mitochondria

Mitochondrial functions are dynamically regulated in the heart. In particular, protein phosphorylation has been shown to be a key mechanism modulating mitochondrial function in diverse cardiovascular phenotypes. However, site-specific phosphorylation information remains scarce for this organ. Accordingly, we performed a comprehensive characterization of murine cardiac mitochondrial phosphoproteome in the context of mitochondrial functional pathways. A platform using the complementary fragmentation technologies of collision-induced dissociation (CID) and electron transfer dissociation (ETD) demonstrated successful identification of a total of 236 phosphorylation sites in the murine heart; 210 of these sites were novel. These 236 sites were mapped to 181 phosphoproteins and 203 phosphopeptides. Among those identified, 45 phosphorylation sites were captured only by CID, whereas 185 phosphorylation sites, including a novel modification on ubiquinol-cytochrome c reductase protein 1 (Ser-212), were identified only by ETD, underscoring the advantage of a combined CID and ETD approach. The biological significance of the cardiac mitochondrial phosphoproteome was evaluated. Our investigations illustrated key regulatory sites in murine cardiac mitochondrial pathways as targets of phosphorylation regulation, including components of the electron transport chain (ETC) complexes and enzymes involved in metabolic pathways (e.g. tricarboxylic acid cycle). Furthermore, calcium overload injured cardiac mitochondrial ETC function, whereas enhanced phosphorylation of ETC via application of phosphatase inhibitors restored calcium-attenuated ETC complex I and complex III activities, demonstrating positive regulation of ETC function by phosphorylation. Moreover, in silico analyses of the identified phosphopeptide motifs illuminated the molecular nature of participating kinases, which included several known mitochondrial kinases (e.g. pyruvate dehydrogenase kinase) as well as kinases whose mitochondrial location was not previously appreciated (e.g. Src). In conclusion, the phosphorylation events defined herein advance our understanding of cardiac mitochondrial biology, facilitating the integration of the still fragmentary knowledge about mitochondrial signaling networks, metabolic pathways, and intrinsic mechanisms of functional regulation in the heart.Mitochondria are the source of energy to sustain life. In addition to their evolutionary origin as an energy-producing organelle, their functionality has integrated into every aspect of life, including the cell cycle, ROS¹ production, apoptosis, and ion balance (1, 2). Our understanding of mitochondrial biology is still growing. Several systems biology approaches have been dedicated to exploring the molecular infrastructure and dynamics of the functional versatility associated with this organelle (3–5).To meet tissue-specific functional demands, mitochondria acquire heterogeneous properties in individual organs, a first statement of their plasticity in function and proteome composition (1, 6). The heterogeneity is evident even in an individual cardiomyocyte (7). A catalogue of the cardiac mitochondrial proteome is emerging via a joint effort (3–5). The dynamics of the mitochondrial proteome manifest at multiple levels, including post-translational modifications, such as phosphorylation. Our investigative goal is to decode this organellar proteome and its post-translational modification in a biological and functional context. In cardiomyocytes, mitochondria are also constantly exposed to fluctuation in energy demands and in ionic conditions. The capacity of mitochondria to cope with such a dynamic environment is essential for the functional role of mitochondria in normal and disease phenotypes (8–10). Unique protein features enabling the mitochondrial proteome to adapt to these biological changes can be interrogated by proteomics tools (10–12). Protein phosphorylation as a rapid and reversible chemical event is an integral component of these protein features (12–14).It has been estimated that one-third of cellular proteins exist in a phosphorylated state at least one time in their lifetime (15). However, only a handful of phosphorylation events have been identified to tune mitochondrial functionality (13, 14, 16) despite the fact that the first demonstration of phosphorylation was reported on a mitochondrial protein more than 5 decades ago (17). Kinases and phosphatases comprise nearly 3% of the human genome (18, 19). In mitochondria, ∼30 kinases and phosphatases have been identified thus far within the expected organellar proteome of a few thousand (3–5, 16). The number of identified mitochondrial phosphoproteins is far below one-third of its proteome size (20). Thus, it appears that the current pool of reported phosphoproteins represents only a small fraction of the anticipated mitochondrial phosphoproteome. The seminal studies from several groups (12–14, 16) demonstrated the prevalence as well as the dynamic nature of phosphorylation in cardiac mitochondria, suggesting that obtaining a comprehensive map of the mitochondrial phosphoproteome is feasible.In this study, we took a systematic approach to tackle the phosphorylation of murine cardiac mitochondrial pathways. We applied the unique strengths of both electron transfer dissociation (ETD) and collision-induced dissociation (CID) LC-MS/MS to screen phosphorylation events in a site-specific fashion. A total of 236 phosphorylation sites in 203 unique phosphopeptides were identified and mapped to 181 phosphoproteins. Novel phosphorylation modifications were discovered in diverse pathways of mitochondrial biology, including ion balance, proteolysis, and apoptosis. Consistent with the role of mitochondria as the major source of energy production under delicate control, metabolic pathways claimed one-third of phosphorylation sites captured in this analysis. To study molecular players steering mitochondrial phosphorylation, we probed the effects of calcium loading on phosphorylation. In addition, a number of kinases with previously unappreciated mitochondrial residence are suggested as potential players modulating mitochondrial pathways. Taken together, the cohort of novel phosphorylation events discovered in this study constitutes an essential step toward the full delineation of the cardiac mitochondrial phosphoproteome.     

Systems-wide Analysis of K-Ras,Cdc42, and PAK4 Signaling by Quantitative Phosphoproteomics

Although K-Ras, Cdc42, and PAK4 signaling are commonly deregulated in cancer, only a few studies have sought to comprehensively examine the spectrum of phosphorylation-mediated signaling downstream of each of these key signaling nodes. In this study, we completed a label-free quantitative analysis of oncogenic K-Ras, activated Cdc42, and PAK4-mediated phosphorylation signaling, and report relative quantitation of 2152 phosphorylated peptides on 1062 proteins. We define the overlap in phosphopeptides regulated by K-Ras, Cdc42, and PAK4, and find that perturbation of these signaling components affects phosphoproteins associated with microtubule depolymerization, cytoskeletal organization, and the cell cycle. These findings provide a resource for future studies to characterize novel targets of oncogenic K-Ras signaling and validate biomarkers of PAK4 inhibition.The Ras oncoproteins are small monomeric GTPases that transduce mitogenic signals from cell surface receptor tyrosine kinases (RTKs) to intracellular serine/threonine kinases. Approximately thirty percent of human tumors harbor a somatic gain-of-function mutation in one of three RAS genes, resulting in the constitutive activation of Ras signaling and the aberrant hyperactivation of growth-promoting effector pathways (1). Designing therapeutic agents that directly target Ras has been challenging (2, 3), and thus clinical development efforts have focused on targeting effector pathways downstream of Ras. The Raf-MEK-ERK and PI3K-Akt effector pathways have been extensively studied and several small molecule inhibitors targeting these pathways are currently under clinical evaluation (4, 5). However, biochemical studies and mouse models indicate that several additional effector pathways are essential for Ras-driven transformation and tumorigenesis (6–11). Hence, a comprehensive characterization of these effector pathways may reveal additional druggable targets.The Rho GTPase Cdc42 lies downstream of Ras (12–14) and regulates many cellular processes that are commonly perturbed in cancer, including migration, polarization, and proliferation (15) (Fig. 1A). Importantly, Cdc42 is overexpressed in several types of human cancer (16–20) and is required for Ras-driven cellular transformation (13, 21, 22). Recent studies show that genetic ablation of Cdc42 impairs Ras-driven tumorigenesis (13), indicating the potential of Cdc42 and its effectors as drug targets in Ras mutant tumors.Open in a separate windowFig. 1.Experimental workflow. A, K-Ras is a small GTPase that regulates the activity of a variety of downstream proteins including the Rho GTPase Cdc42. The PAK4 serine/threonine kinase is a direct effector of Cdc42 and regulates actin reorganization, microtubule stability, and cell polarity. B, To measure large-scale phosphorylation changes induced by constitutive K-Ras or Cdc42 signaling or PAK4 ablation, the quantitative label-free PTMscan® approach was employed (Cell Signaling Technology). Briefly, for each condition extracted proteins were digested with trypsin and separated from non-peptide material by solid-phase extraction with Sep-Pak C18 cartridges. Three phosphorylation motif antibodies were used serially to isolate phosphorylated peptides in independent immunoaffinity purifications (CDK substrate motif [K R]-pS-P-X-[K R], CK substrate motif pT-[D E]-X-[D E], PKD substrate motif l-X-R-X-X-p[S T]). The samples were run in duplicate and tandem mass spectra were collected with an LTQ-Orbitrap hybrid mass spectrometer. pLPC is an empty vector control.In particular, the p21-activated kinases (PAKs) are Cdc42 effectors that have generated significant interest (23, 24), as they are central components of key oncogenic signaling pathways and regulate cytoskeletal organization, cell migration, and nuclear signaling (25). The PAK family is comprised of six members and is subdivided into two groups (Groups I and II) based on sequence and structural homology. Group I PAKs (PAK1–3) are relatively well characterized, however, much less is known regarding the function and regulation of Group II PAKs (PAK4–6). The kinase domains of Group I and II PAKs share only about 50% identity, suggesting the two groups may recognize distinct substrates and govern unique cellular processes (26).The Group II PAK family member PAK4 is of particular interest as it is overexpressed or genetically amplified in several lung, colon, prostate, pancreas, and breast tumor cell lines and samples (26–30). Furthermore, functional studies have implicated PAK4 in cell transformation, cell invasion, and migration (27, 31). Xenograft studies in athymic mice show an important role for PAK4 in mediating Cdc42- or K-Ras-driven tumor formation, highlighting a critical role for Pak4 downstream of these GTPases (32). Given its roles in transformation, tumorigenesis, and oncogenic signaling, there is significant interest in targeting PAK4 therapeutically (23). PAK4 binds and phosphorylates several proteins involved in cytoskeletal organization and apoptosis, including Lim domain kinase 1 (LIMK1) (33), guanine nucleotide exchange factor-H1 (GEF-H1) (34), Raf-1 (35), and Bad (36). However, the Group I PAK family member PAK1 also phosphorylates several of these PAK4 targets (37). Thus, there remains a need to identify robust and selective pharmacodynamic biomarkers for PAK4 inhibition.Despite the importance of PAK4 and its upstream regulators in cancer development, few studies have sought to comprehensively characterize the spectrum of K-Ras, Cdc42, or PAK4 mediated phosphorylation signaling (37–39). Recent developments in mass spectrometry allow the in-depth identification and quantitation of thousands of phosphorylation sites (40–43). The majority of large-scale efforts have aimed to identify the basal phosphoproteomes of different species (44, 45) or tissues (46) to characterize global steady-state phosphorylation. However, this methodology can also be applied to quantify perturbed phosphorylation regulation in cancer signaling pathways (40, 47–49), and has the potential to reveal novel biomarkers of oncogenic signaling.In this study, we completed a label-free quantitative analysis of K-Ras, Cdc42, and PAK4 phosphorylation signaling using the PTMScan® method, which has proven as robust and reproducible quantitation technology (50, 51). We quantified phosphorylation levels in wild-type and PAK4 knockout NIH3T3 cells expressing oncogenic K-Ras, activated Cdc42, or an empty vector control to elucidate the molecular pathways and functions modulated by these key signaling proteins. We report relative quantitation of 2152 phosphorylated peptides on 1062 proteins among the different conditions, and find that many of the regulated phosphoproteins are associated with microtubule depolymerization, cytoskeletal organization, and the cell cycle. To our knowledge, our study is the first to examine the overlap among signaling networks regulated by K-Ras, Cdc42, and PAK4, and provides a resource for future studies to further interrogate the perturbation of this signaling pathway.     

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.