Identification of a novel phosphorylation site in protein phosphatase inhibitor-1 as a negative regulator of cardiac function

Human and experimental heart failure is characterized by increases in type-1 protein phosphatase activity, which may be partially attributed to inactivation of its endogenous regulator, protein phosphatase inhibitor-1. Inhibitor-1 represents a nodal integrator of two major second messenger pathways, adenosine 3',5'-cyclic monophosphate (cAMP) and calcium, which mediate its phosphorylation at threonine 35 and serine 67, respectively. Here, using recombinant inhibitor-1 wild-type and mutated proteins, we identified a novel phosphorylation site in inhibitor-1, threonine 75. This phosphoamino acid was phosphorylated in vitro by protein kinase Calpha independently and to the same extent as serine 67, the previous protein kinase Calpha-identified site. Generation of specific antibodies for the phosphorylated and dephosphorylated threonine 75 revealed that this site is phosphorylated in rat and dog hearts. Adenoviral-mediated expression of the constitutively phosphorylated threonine 75 inhibitor-1 in isolated myocytes was associated with specific stimulation of type-1 protein phosphatase activity and marked inhibition of the sarcoplasmic calcium pump affinity for calcium, resulting in depressed contractility. Thus, phosphorylation of inhibitor-1 at threonine 75 represents a new mechanism of cardiac contractility regulation, partially through the alteration of sarcoplasmic reticulum calcium transport activity.     

Identification of Ser-543 as the major regulatory phosphorylation site in spinach leaf nitrate reductase.

Spinach leaf NADH:nitrate reductase (NR) responds to light/dark signals and photosynthetic activity in part as a result of rapid regulation by reversible protein phosphorylation. We have identified the major regulatory phosphorylation site as Ser-543, which is located in the hinge 1 region connecting the cytochrome b domain with the molybdenum-pterin cofactor binding domain of NR, using recombinant NR fragments containing or lacking the phosphorylation site sequence. Studies with NR partial reactions indicated that the block in electron flow caused by phosphorylation also could be localized to the hinge 1 region. A synthetic peptide (NR6) based on the phosphorylation site sequence was phosphorylated readily by NR kinase (NRk) in vitro. NR6 kinase activity tracked the ATP-dependent inactivation of NR during several chromatographic steps and completely inhibited inactivation/phosphorylation of native NR in vitro. Two forms of NRk were resolved by using anion exchange chromatography. Studies with synthetic peptide analogs indicated that both forms of NRk had similar specificity determinants, requiring a basic residue at P-3 (i.e., three amino acids N-terminal to the phosphorylated serine) and a hydrophobic residue at P-5. Both forms are strictly calcium dependent but belong to distinct families of protein kinases because they are distinct immunochemically.     

Identification of a novel phosphorylation site in ataxin-1

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disease resulting from an expanded CAG repeat in the SCA1 gene that leads to an expanded polyglutamine tract in the gene product. Previous studies have demonstrated that serine at site 776 is phosphorylated [E.S. Emiamian, M.D. Kaytor, L.A. Duvick, T. Zu, S.K. Tousey, H.Y. Zoghbi, H.B. Clark, H.T. Orr, Serine 776 of ataxin-1 is critical for polyglutamine-induced disease in SCA1 transgenic mice, Neuron 38 (2003) 375-387.]. Studies of ataxin-1 S776 and serine mutated to an alanine, A776, have also shown differential protein-protein interactions and reduced neurodegeneration [H.K. Chen, P. Fernandez-Funez, S.F. Acevedo, Y.C. Lam, M.D. Kaytor, M.H. Fernandez, A. Aitken, E.M. Skoulakis, H.T. Orr, J. Botas, H.Y. Zoghbi, Interaction of Akt_phosphorylated ataxin-1 with 14-3-3 mediates neurodegeneration in spinocerebellar ataxia type 1.]. However, mutation of the site serine 776 to an alanine did not abolish all phosphorylation of the protein ataxin-1, suggesting the presence of additional phosphorylation sites [E.S. Emiamian, M.D. Kaytor, L.A. Duvick, T. Zu, S.K. Tousey, H.Y. Zoghbi, H.B. Clark, H.T. Orr, Serine 776 of ataxin-1 is critical for polyglutamine-induced disease in SCA1 transgenic mice, Neuron 38 (2003) 375-387.]. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and mutational analysis demonstrated a novel phosphorylation site at serine 239 of ataxin-1.     

Identification of Ser-55 as a major protein kinase A phosphorylation site on the 70-kDa subunit of neurofilaments. Early turnover during axonal transport.

The 70-kDa neurofilament protein subunit (NF-L) is phosphorylated in vivo on at least three sites (L1 to L3) (Sihag, R. K. and Nixon, R. A. (1989) J. Biol. Chem. 264, 457-464). The turnover of phosphate groups on NF-L during axonal transport was determined after the neurofilaments in retinal ganglion cells were phosphorylated in vivo by injecting mice intravitreally with [32P]orthophosphate. Two-dimensional phosphopeptide maps of NF-L from optic axons of mice 10 to 90 h after injection showed that radiolabel decreased faster from peptides L2 and L3 than from L1 as neurofilaments were transported. To identify phosphorylation sites on peptide L2, axonal cytoskeletons were phosphorylated by protein kinase A in the presence of heparin. After the isolated NF-L subunits were digested with alpha-chymotrypsin, 32P-peptides were separated by high performance liquid chromatography on a reverse-phase C8 column. Two-dimensional peptide mapping showed that the alpha-chymotrypsin 32P-peptide accepting most of the phosphates from protein kinase A migrated identically with the in vivo-labeled phosphopeptide L2. The sequence of this peptide (S-V-R-R-S-Y) analyzed by automated Edman degradation corresponded to amino acid residues 51-56 of the NF-L sequence. A synthetic 13-mer (S-L-S-V-R-R-S-Y-S-S-S-S-G) corresponding to amino acid residues 49-61 of NF-L was also phosphorylated by protein kinase A. alpha-Chymotryptic digestion of the 13-mer generated a peptide which contained most of the phosphates and co-migrated with the phosphopeptide L2 on two-dimensional phosphopeptide maps. Edman degradation of the phosphorylated 13-mer identified serine residue 55 which is located within a consensus phosphorylation sequence for protein kinase A as the major site of phosphorylation. Since protein kinase A-mediated phosphorylation influences intermediate filament assembly/disassembly in vitro, we propose that the phosphopeptide L2 region is a neurofilament-assembly domain and that the cycle of phosphorylation and dephosphorylation of Ser-55 on NF-L, which occurs relatively early after subunit synthesis in vivo, regulaaes a step in neurofilament assembly or initial interactions during axonal transport.     

Identification of the site of phosphorylation of the chemotaxis response regulator protein, CheY

The protein (Escherichia coli CheY) that controls the direction of flagellar rotation during bacterial chemotaxis has been shown to be phosphorylated on the aspartate 57 residue. The residue phosphorylated is present within a conserved sequence in every member of a family of bacterial regulatory proteins. The phosphorylation is transient, with a much shorter half-life than that expected of a simple acyl phosphate intermediate, indicating that the sequence and conformation of the protein is designed to achieve a rapid hydrolysis. The CheY-phosphate linkage can be reductively cleaved by sodium borohydride. High-performance tandem mass-spectrometric analysis of proteolytic peptides derived from [3H]borohydride-reduced phosphorylated CheY protein was used to identify the position of phosphorylation. Mutants with altered aspartate 57 exhibited no chemotaxis. When aspartate 13, another conserved residue, was changed, greatly reduced chemotaxis was observed, suggesting an important role for aspartate 13. The rate-determining step of chemotactic signaling is governed by the kinetics of formation and hydrolysis of the CheY protein phosphoaspartate bond. The CheY protein apparently functions as a protein phosphatase that possesses a transient covalent intermediate. Transient phosphorylation of an aspartate residue is an effective mechanism for producing a biochemical signal with a short concentration-independent half-life. The duration of the signal can be controlled by small structural elements within the phosphorylated protein.     

Identification of a novel phosphorylation site in human androgen receptor by mass spectrometry

An N-terminal hexahistidine-tagged full-length human androgen receptor protein (His(6)-hAR) was overexpressed and purified to apparent homogeneity in the presence of dihydrotestosterone (DHT) in our previous studies. In-gel trypsin digestion of the purified DHT-bound His(6)-hAR, and tryptic peptide mapping using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS), detected a total of 17 peptides (21% coverage of hAR) with 9 peptides originating from the ligand-binding domain (LBD, 31% coverage of LBD). Amino acid sequencing analysis of the tryptic peptides from a separate in-gel digestion of the His(6)-hAR, using HPLC-coupled electrospray ionization ion trap mass spectrometry (LC/ESI-ITMS and MS/MS), unambiguously confirmed 21 peptides with 19% coverage of the hAR, of which 11 peptides originated from the LBD (35% coverage of LBD). These 21 peptides included 11 out of the 17 peptides detected by MALDI/TOF-MS. In addition, a novel serine phosphorylation site (Ser(308)) within the N-terminal transactivation domain of hAR was identified.     

Identification of a novel phosphorylation site on histone H3 coupled with mitotic chromosome condensation.

Histone H3 (H3) phosphorylation at Ser(10) occurs during mitosis in eukaryotes and was recently shown to play an important role in chromosome condensation in Tetrahymena. When producing monoclonal antibodies that recognize glial fibrillary acidic protein phosphorylation at Thr(7), we obtained some monoclonal antibodies that cross-reacted with early mitotic chromosomes. They reacted with 15-kDa phosphoprotein specifically in mitotic cell lysate. With microsequencing, this phosphoprotein was proved to be H3. Mutational analysis revealed that they recognized H3 Ser(28) phosphorylation. Then we produced a monoclonal antibody, HTA28, using a phosphopeptide corresponding to phosphorylated H3 Ser(28). This antibody specifically recognized the phosphorylation of H3 Ser(28) but not that of glial fibrillary acidic protein Thr(7). Immunocytochemical studies with HTA28 revealed that Ser(28) phosphorylation occurred in chromosomes predominantly during early mitosis and coincided with the initiation of mitotic chromosome condensation. Biochemical analyses using (32)P-labeled mitotic cells also confirmed that H3 is phosphorylated at Ser(28) during early mitosis. In addition, we found that H3 is phosphorylated at Ser(28) as well as Ser(10) when premature chromosome condensation was induced in tsBN2 cells. These observations suggest that H3 phosphorylation at Ser(28), together with Ser(10), is a conserved event and is likely to be involved in mitotic chromosome condensation.     

Identification of a CK2 phosphorylation site in mdm2.

Mdm2 is a cellular oncoprotein the most obvious function of which is the down-regulation of the growth suppressor protein p53. It represents a highly phosphorylated protein but only little is yet known about the sites phosphorylated in vivo, the kinases that are responsible for the phosphorylation or the functional relevance of the phosphorylation status. Recently, we have shown that mdm2 is a good substrate for protein kinase CK2 at least in vitro. Computer analysis of the primary amino acid sequence of mdm2 revealed 19 putative CK2 phosphorylation sites. By using deletion mutants of mdm2 and a peptide library we identified the serine residue at position 269 which lies within a canonical CK2 consensus sequence (EGQELSDEDDE) as the most important CK2 phosphorylation site. Moreover, by using the mdm2 S269A mutant for in vitro phosphorylation assays this site was shown to be phosphorylated by CK2. Binding studies revealed that phosphorylation of mdm2 at S269 does not have any influence on the binding of p53 to mdm2.     

Identification of a central phosphorylation site in p21-activated kinase regulating autoinhibition and kinase activity.

p21-activated kinases (Pak)/Ste20 kinases are regulated in vitro and in vivo by the small GTP-binding proteins Rac and Cdc42 and lipids, such as sphingosine, which stimulate autophosphorylation and phosphorylation of exogenous substrates. The mechanism of Pak activation by these agents remains unclear. We investigated Pak kinase activation in more detail to gain insight into the interplay between the GTPase/sphingosine binding, an intramolecular inhibitory interaction, and autophosphorylation. We present biochemical evidence that an autoinhibitory domain (ID) contained within amino acid residues 67-150 of Pak1 interacts with the carboxyl-terminal kinase domain and that this interaction is regulated in a GTPase-dependent fashion. Cdc42- and sphingosine-stimulated Pak1 activity can be inhibited in trans by recombinant ID peptide, indicating similarities in their mode of activation. However, Pak1, which was autophosphorylated in response to either GTPase or sphingosine, is highly active and is insensitive to inhibition by the ID peptide. We identified phospho-acceptor site threonine 423 in the kinase activation loop as a critical determinant for the sensitivity to autoinhibition and enzymatic activity. Phosphorylation studies suggested that the stimulatory effect of both GTPase and sphingosine results in exposure of the activation loop, making it accessible for intermolecular phosphorylation.     

Activation of CheY mutant D57N by phosphorylation at an alternative site, Ser-56

The site of phosphorylation of the chemotaxis response regulator CheY is aspartate 57. When Asp-57 is replaced with an asparagine, the resultant protein can be phosphorylated at an alternative site. We report here that phosphorylation of this mutant protein, CheY D57N, at the alternative site affords the protein activity in vivo in the absence of CheZ. Using a direct phosphopeptide mapping approach, we identified the alternate phosphorylation site as serine 56. Introduction of a Ser-->Ala substitution at this position in wild-type CheY had no effect on function. However, replacement of Ser-56 with Ala in CheY D57N abrogated the activity seen in vivo for the CheY D57N single mutant protein, and no phosphorylation of the CheY S56A/D57N double mutant protein was observed in vitro. Construction and analysis of double mutants CheY D57N/T87A and CheY D57N/K109R, which were both inactive, suggested that phosphorylation at Ser-56 or Asp-57 may activate the protein by similar mechanisms. In contrast to CheY D57N, mutant CheY D57E displayed no activity in vivo, despite its ability to be phosphorylated in vitro. Acid-base stability analysis indicated that CheY D57E phosphorylates on an acidic residue, presumably Glu-57. These data suggest that a key determinant of the ability of a phosphoryl group to activate CheY is proximity to the hydrophobic core of the protein, with consequent opportunity to reposition key residues, irrespective of the chemical nature of the linkage attaching the phosphoryl group to CheY.     

Identification and characterization of PS-GAP as a novel regulator of caspase-activated PAK-2

p21-activated protein kinase (PAK)-2 is a member of the PAK family of serine/threonine kinases. PAKs are activated by the p21 G-proteins Rac and Cdc42 in response to a variety of extracellular signals and act in pathways controlling cell growth, shape, motility, survival, and death. PAK-2 is unique among the PAK family members because it is also activated through proteolytic cleavage by caspase-3 or similar proteases to generate the constitutively active PAK-2p34 fragment. Activation of full-length PAK-2 by Rac or Cdc42 stimulates cell survival and protects cells from cell death, whereas caspase-activated PAK-2p34 induces a cell death response. Caspase-activated PAK-2p34 is rapidly degraded by the 26 S proteasome, but full-length PAK-2 is not. Stabilization of PAK-2p34 by preventing its polyubiquitination and degradation results in a dramatic stimulation of cell death. Although many proteins have been shown to interact with and regulate full-length PAK-2, little is known about the regulation of caspase-activated PAK-2p34. Here, we identify PS-GAP as a regulator of caspase-activated PAK-2p34. PS-GAP is a GTPase-activating protein for Cdc42 and RhoA that was originally identified by its interaction with the tyrosine kinase PYK-2. PS-GAP interacts specifically with caspase-activated PAK-2p34, but not active or inactive full-length PAK-2, through a region between the GAP and SH3 domains. The interaction with PS-GAP inhibits the protein kinase activity of PAK-2p34 and changes the localization of PAK-2p34 from the nucleus to the perinuclear region. Furthermore, PS-GAP decreases the stimulation of cell death induced by stabilization of PAK-2p34.     

Identification of a new tamoxifen-xanthene hybrid as pro-apoptotic anticancer agent

Breast cancer is the most diagnosed type of cancer among women for which an exhaustive cure has not been discovered yet. Nowadays, tamoxifen still represents the gold standard for breast cancer therapy; it acts on both estrogen receptor-positive and estrogen receptor-negative breast cancers. Unfortunately, its toxicity and the related chemoresistance undermine its antitumor potential. In this paper, new tamoxifen-based derivatives with a rigid structural motif in their structure were designed, synthesized, and evaluated to assess their antitumor behavior. All the tested compounds affected estrogen receptor-positive tumor (MCF-7) cell growth, even with different extents, among which, the most active ones proved also to induce mitochondria-mediated apoptosis through activation of PARP cleavage, decrease in Bax/Bcl-2 ratio and increase in Bim gene expression levels. Here we found that the compound 1, carrying a rigid xanthene core, turned out to be the most promising of the set showing an activity profile comparable to that of tamoxifen. Furthermore, a more favorable genotoxic profile than tamoxifen made compound 1 a promising candidate for further studies.     

Identification of in vivo phosphorylation sites on human deoxycytidine kinase. Role of Ser-74 in the control of enzyme activity

Deoxycytidine kinase (dCK) catalyzes the rate-limiting step of the deoxyribonucleoside salvage pathway in mammalian cells and plays a key role in the activation of numerous nucleoside analogues used in anti-cancer and antiviral chemotherapy. Although compelling evidence indicated that dCK activity might be regulated by phosphorylation/dephosphorylation, direct demonstration was lacking. Here we showed that dCK overexpressed in HEK 293T cells was labeled after incubating the cells with [32P]orthophosphate. Sorbitol, which was reported to decrease dCK activity, also decreased the labeling of dCK. These results indicated that dCK may exist as a phosphoprotein in vivo and that its activity can be correlated with its phosphorylation level. After purification of 32P-labeled dCK, digestion by trypsin, and analysis of the radioactive peptides by tandem mass spectrometry, the following four in vivo phosphorylation sites were identified: Thr-3, Ser-11, Ser-15, and Ser-74, the latter being the major phosphorylation site. Site-directed mutagenesis and use of an anti-phospho-Ser-74 antibody demonstrated that Ser-74 phosphorylation was crucial for dCK activity in HEK 293T cells, whereas phosphorylation of other identified sites did not seem essential. Phosphorylation of Ser-74 was also detected on endogenous dCK in leukemic cells, in which the Ser-74 phosphorylation state was increased by agents that enhanced dCK activity. Our study provided direct evidence that dCK activity can be controlled by phosphorylation in intact cells and highlights the importance of Ser-74 for dCK activity.     

Identification of a urokinase receptor-integrin interaction site. Promiscuous regulator of integrin function

Adhesion and signaling by integrins require their dynamic association with nonintegrin membrane proteins. One such protein, the glycolipid-anchored urokinase receptor (uPAR), associates with and modifies the function of the beta(2)-integrin Mac-1 (CD11b/CD18). In this study, a critical non-I-domain binding site for uPAR on CD11b (M25; residues 424-440) is identified by homology with a phage display peptide known to bind uPAR. Recombinant soluble uPAR and cells expressing uPAR bound to immobilized M25, binding being promoted by urokinase and blocked by soluble M25, but not a scrambled control or homologous peptides from other beta(2)-associated alpha-chains. Mac-1, but not a mutated Mac-1 in which M25 was replaced with the homologous sequence of CD11c, co-precipitated with uPAR. In the beta-propeller model of alpha-chain folding, M25 spans an exposed loop on the ligand-binding, upper surface of alphaM, identifying uPAR as an atypical alphaM ligand. Although not blocking ligand binding to Mac-1, M25 (25-100 microM) inhibited leukocyte adhesion to fibrinogen, vitronectin, and cytokine-stimulated endothelial cells. M25 also blocked the association of uPAR with beta(1)-integrins and impaired beta(1)-integrin-dependent spreading and migration of human vascular smooth muscle cells on fibronectin and collagen. These observations indicate that uPAR associates with integrins directly and that disruption of this association broadly impairs integrin function, suggesting a novel strategy for regulation of integrins in the settings of inflammation and tumor progression.     

Identification of Lys-Pro-Gln as a novel cleavage site specificity of saliva-associated proteases

The nonsterile environment of the oral cavity facilitates substantial proteolytic processing, not only of resident salivary proteins but also of dietary proteins. To gain insight into whole saliva enzymatic processes, the in vivo generated peptides in this oral fluid were subjected to nano-flow liquid chromatography electrospray ionization tandem mass spectrometry. The 182 peptides identified were predominantly derived from acidic and basic proline-rich proteins, statherin, and histatins. The proteolytic cleavages in the basic proline-rich proteins occurred preferentially after a Gln residue with predominant specificity for the tripeptide Xaa-Pro-Gln, where Xaa in the P(3) position was mostly represented by Lys. Using the synthetic substrates Lys-Pro-Gln-pNA and Gly-Gly-Gln-pNA, the overall K(m) values were determined to be 97 +/- 7.7 and 611 +/- 28 microm, respectively, confirming glutamine endoprotease activity in whole saliva and the influence of the amino acids in positions P(2) and P(3) on protease recognition. The pH optimum of Lys-Pro-Gln-pNA hydrolysis was 7.0, and the activity was most effectively inhibited by antipain and 4-(2-aminoethyl) benzenesulfonyl fluoride, was metal ion-dependent, and not inhibited by cysteine protease inhibitors. A systematic evaluation of enzyme activities in various exocrine and nonexocrine contributors to whole saliva revealed that the glutamine endoprotease is derived from dental plaque and likely microbial in origin. The P(1) site being occupied by a Gln residue is a nonarchetype with respect to known proteases and indicates the presence of novel glutamine-specific endoprotease(s) in oral fluid.     

Identification of a novel Bcl-xL phosphorylation site regulating the sensitivity of taxol- or 2-methoxyestradiol-induced apoptosis

Bcl-xL, a close homolog of Bcl2, is an important regulator of apoptosis and is overexpressed in human cancer. Phosphorylation of Bcl-xL can be induced by microtubule-damaging drugs such as taxol or 2-methoxyestradiol (2-ME). By site-directed mutagenesis studies, we have identified that serine 62 is the necessary site for taxol- or 2-ME-induced Bcl-xL phosphorylation in prostate cancer cells. Further studies with the inhibitor of Jun kinase (JNK) and phosphorylation null mutant of Bcl-xL reveal the augmentative role of JNK-mediated Bcl-xL phosphorylation in apoptosis of prostate cancer cells. In summary, our studies suggest that the phosphorylation of Bcl-xL by stress response kinase signaling might oppose the anti-apoptotic function of Bcl-xL to permit prostate cancer cells to die by apoptosis.     

Identification of tyrosine 972 as a novel site of Jak2 tyrosine kinase phosphorylation and its role in Jak2 activation

Jak2 is a 130 kDa tyrosine kinase that is important in a number of cellular signaling pathways. Its function is intrinsically regulated by the phosphorylation of a handful of its 49 tyrosines. Here, we report that tyrosine 972 (Y972) is a novel site of Jak2 phosphorylation and, hence, autoregulation. Specifically, we found that Y972 is phosphorylated and confirmed that this residue resides on the surface of the protein. Using expression plasmids that expressed either wild-type Jak2 or a full-length Jak2 cDNA containing a single Y972F substitution mutation, we investigated the consequences of losing Y972 phosphorylation on Jak2 function. We determined that the loss of Y972 phosphorylation significantly reduced the levels of both Jak2 total tyrosine phosphorylation and phosphorylation of Y1007/Y1008. Additionally, Y972 phosphorylation was shown to be important for maximal kinase function. Interestingly, in response to classical cytokine activation, the Jak2 Y972F mutant exhibited a moderately impaired level of activation when compared to the wild-type protein. However, when Jak2 was activated via a GPCR ligand, the ability of the Y972F mutant to be activated was completely lost, therefore suggesting a differential role of Y972 in Jak2 activation. Finally, we found that phosphorylation of Y972 enhances Jak2 kinase function via a mechanism that appears to stabilize the active conformation of the protein. Collectively, our results suggest that Y972 is a novel site of Jak2 phosphorylation and plays an important differential role in ligand-dependent Jak2 activation via a mechanism that involves stabilization of the Jak2 active conformation.