Primary Phosphorylation Site Research Articles

Expression and characterization of a low molecular weight recombinant human gelatin: development of a substitute for animal-derived gelatin with superior features

Gelatin is used as a stabilizer in several vaccines. Allergic reactions to gelatins have been reported, including anaphylaxis. These gelatins are derived from animal tissues and thus represent a potential source of contaminants that cause transmissible spongiform encephalopathies. We have developed a low molecular weight human sequence gelatin that can substitute for the animal sourced materials. A cDNA fragment encoding 101 amino acids of the human proα1 (I) chain was amplified, cloned into plasmid pPICZα, integrated into Pichia pastoris strain X-33, and isolates expressing high levels of recombinant gelatin FG-5001 were identified. Purified FG-5001 was able to stabilize a live attenuated viral vaccine as effectively as porcine gelatin. This prototype recombinant gelatin was homogeneous with respect to molecular weight but consisted of several charge isoforms. These isoforms were separated by cation exchange chromatography and found to result from a combination of truncation of the C-terminal arginine and post-translational phosphorylation. Site-directed mutagenesis was used to identify the primary site of phosphorylation as serine residue 546; serine 543 was phosphorylated at a low level. A new construct was designed encoding an engineered gelatin, FG-5009, with point mutations that eliminated the charge heterogeneity. FG-5009 was not recognized by antigelatin IgE antibodies from children with confirmed gelatin allergies, establishing the low allergenic potential of this gelatin. The homogeneity of FG-5009, the ability to produce large quantities in a reproducible manner, and its low allergenic potential make this a superior substitute for the animal gelatin hydrolysates currently used to stabilize many pharmaceuticals.

Phosphorylation of NG2 Proteoglycan by Protein Kinase C-α Regulates Polarized Membrane Distribution and Cell Motility

Protein kinase C (PKC)-alpha phosphorylation of recombinant NG2 cytoplasmic domain and phorbol ester-induced PKC-dependent phosphorylation of full-length NG2 expressed in U251 cells are both blocked by mutation of Thr(2256), identifying this residue as a primary phosphorylation site. In untreated U251/NG2 cells, NG2 is present along with ezrin and alpha(3)beta(1) integrin in apical cell surface protrusions. Phorbol ester treatment causes redistribution of all three components to lamellipodia, accompanied by increased cell motility. U251 cells expressing NG2 with a valine substitution at position 2256 are resistant to phorbol ester treatment: NG2 remains in membrane protrusions and cell motility is unchanged. In contrast, NG2 with a glutamic acid substitution at position 2256 redistributes to lamellipodia even without phorbol ester treatment, rendering transfected U251 cells spontaneously motile. PKC-alpha-mediated NG2 phosphorylation at Thr(2256) is therefore a key step for initiating cell polarization and motility.

Smooth Muscle Phosphatase Is Regulated in Vivo by Exclusion of Phosphorylation of Threonine 696 of MYPT1 by Phosphorylation of Serine 695 in Response to Cyclic Nucleotides

Regulation of smooth muscle myosin phosphatase (SMPP-1M) is thought to be a primary mechanism for explaining Ca(2+) sensitization/desensitization in smooth muscle. Ca(2+) sensitization induced by activation of G protein-coupled receptors acting through RhoA involves phosphorylation of Thr-696 (of the human isoform) of the myosin targeting subunit (MYPT1) of SMPP-1M inhibiting activity. In contrast, agonists that elevate intracellular cGMP and cAMP promote Ca(2+) desensitization in smooth muscle through apparent activation of SMPP-1M. We show that cGMP-dependent protein kinase (PKG)/cAMP-dependent protein kinase (PKA) efficiently phosphorylates MYPT1 in vitro at Ser-692, Ser-695, and Ser-852 (numbering for human isoform). Although phosphorylation of MYPT1 by PKA/PKG has no direct effect on SMPP-1M activity, a primary site of phosphorylation is Ser-695, which is immediately adjacent to the inactivating Thr-696. In vitro, phosphorylation of Ser-695 by PKA/PKG appeared to prevent phosphorylation of Thr-696 by MYPT1K. In ileum smooth muscle, Ser-695 showed a 3-fold increase in phosphorylation in response to 8-bromo-cGMP. Addition of constitutively active recombinant MYPT1K to permeabilized smooth muscles caused phosphorylation of Thr-696 and Ca(2+) sensitization; however, this phosphorylation was blocked by preincubation with 8-bromo-cGMP. These findings suggest a mechanism of Ca(2+) desensitization in smooth muscle that involves mutual exclusion of phosphorylation, whereby phosphorylation of Ser-695 prevents phosphorylation of Thr-696 and therefore inhibition of SMPP-1M.

The phosphorylation of caveolin-2 on serines 23 and 36 modulates caveolin-1-dependent caveolae formation.

Caveolin-1 and -2 are the two major coat proteins found in plasma membrane caveolae of most of cell types. Here, by using adenoviral transduction of either caveolin-1 or caveolin-2 or both isoforms into cells lacking both caveolins, we demonstrate that caveolin-2 positively regulates caveolin-1-dependent caveolae formation. More importantly, we show that caveolin-2 is phosphorylated in vivo at two serine residues and that the phosphorylation of caveolin-2 is necessary for its actions as a positive regulator of caveolin-1 during organelle biogenesis in prostate cancer cells. Mutation of the primary phosphorylation sites on caveolin-2, serine 23 and 36, reduces the number of plasmalemma-attached caveolae and increases the accumulation of noncoated vesicles, but does not affect trafficking of caveolin-2, interaction with caveolin-1 or its biophysical properties. Thus, the phosphorylation of caveolin-2 is a novel mechanism to regulate the dynamics of caveolae assembly.

Hedgehog-stimulated Phosphorylation of the Kinesin-related Protein Costal2 Is Mediated by the Serine/Threonine Kinase Fused

The Hedgehog (Hh) signaling molecule is required for the development of numerous tissues in Drosophila. Within the cell, Hh signal transduction utilizes a large protein complex consisting of the Fused (Fu), Costal2 (Cos2), and Cubitis interruptus (Ci) proteins, but the functional interactions between these proteins are still largely uncharacterized. Using a baculovirus system, we demonstrate that the serine/threonine kinase Fu phosphorylates the kinesin-like protein Cos2 when coexpressed with Cos2. Coexpression of Cos2 and a kinase-inactive version of Fu eliminates the majority of Cos2 phosphorylation. We then show that the primary Fu-induced phosphorylation site of Cos2 is serine 572, whereas serine 931 is phosphorylated to a lesser extent. Mutation of serine 572 to alanine eliminates most, but not all, specific phosphopeptides of Cos2 when coexpressed with Fu. We also demonstrate that the phosphorylation pattern of Cos2 produced by baculovirus coexpression with kinase-dead Fu is almost identical to the phosphorylation pattern of Cos2 isolated from unstimulated S2 cells. Finally, the phosphorylation pattern of Cos2 produced by baculovirus coinfection with wild-type Fu is almost identical to that of Cos2 isolated from S2 cells stimulated by Hh, indicating that phosphorylation of serines 572 and 931 is a genuine Hh signaling event. This study clarifies the unique functions of Fu and Cos2 in Hh signal transduction and identifies only the second known phosphorylation site of a kinesin-like molecule.

Platelet-derived Growth Factor (PDGF)-induced Tyrosine Phosphorylation of the Low Density Lipoprotein Receptor-related Protein (LRP): EVIDENCE FOR INTEGRATED CO-RECEPTOR FUNCTION BETWEEN LRP AND THE PDGF

The low density lipoprotein receptor-related protein (LRP) functions in the catabolism of numerous ligands including proteinases, proteinase inhibitor complexes, and lipoproteins. In the current study we provide evidence indicating an expanded role for LRP in modulating cellular signaling events. Our results show that platelet-derived growth factor (PDGF) BB induces a transient tyrosine phosphorylation of the LRP cytoplasmic domain in a process dependent on PDGF receptor activation and c-Src family kinase activity. Other growth factors, including basic fibroblast growth factor, epidermal growth factor, insulin-like growth factor-1, were unable to mediate tyrosine phosphorylation of LRP. The basis for this selectivity may result from the ability of LRP to bind PDGFBB, because surface plasmon resonance experiments demonstrated that only PDGF, and not basic fibroblast growth factor, epidermal growth factor, or insulin-like growth factor-1, bound to purified LRP immobilized on a sensor chip. The use of LRP mini-receptor mutants as well as in vitro phosphorylation studies demonstrated that the tyrosine located within the second NPXY motif found in the LRP cytoplasmic domain is the primary site of tyrosine phosphorylation by Src and Src family kinases. Co-immunoprecipitation experiments revealed that PDGF-mediated tyrosine phosphorylation of LRPs cytoplasmic domain results in increased association of the adaptor protein Shc with LRP and that Shc recognizes the second NPXY motif within LRPs cytoplasmic domain. In the accompanying paper, Boucher et al. (Boucher, P., Liu, P. V., Gotthardt, M., Hiesberger, T., Anderson, R. G. W., and Herz, J. (2002) J. Biol. Chem. 275, 15507-15513) reveal that LRP is found in caveolae along with the PDGF receptor. Together, these studies suggest that LRP functions as a co-receptor that modulates signal transduction pathways initiated by the PDGF receptor.

Protein kinase C mediates phosphorylation of the regulatory light chain of myosin-II during mitosis.

Phosphorylation of the regulatory light chain (RLC) of myosin-II is cell cycle dependent. Early in mitosis the RLC is phosphorylated predominantly on Ser-1/2, while during cytokinesis the primary site of phosphorylation is Ser-19 (Yamakita et al., 1994). To identify candidate kinases likely to mediate the mitotic phosphorylation on Ser-1/2, we assayed RLC kinase activity in mitotic cell extracts and measured apparent steady-state kinetic constants using purified enzymes. The mitotic RLC kinase is distinct from cdc2 kinase, protein kinase A and protein kinase G, as activators or inhibitors specific for these kinases do not affect the mitotic kinase activity. The activity of the mitotic RLC kinase is enhanced by the addition of Ca2+ and DAG and/or phorbol esters, characteristics of a conventional protein kinase C (PKC). Moreover, the PKC inhibitors, Gö6983 and Gö6976, significantly attenuate the phosphorylation of the RLC in mitotic extracts. Apparent steady-state kinetic studies indicate that several PKC isoforms display high specificity for myosin-II. These results suggest that current models describing Ser-1/2 phosphorylation during mitosis need to be re-evaluated.

Hierarchical Phosphorylation of δ-Opioid Receptor Regulates Agonist-induced Receptor Desensitization and Internalization

Treatment of HEK293 cells expressing the delta-opioid receptor with agonist [d-Pen(2,5)]enkephalin (DPDPE) resulted in the rapid phosphorylation of the receptor. We constructed several mutants of the potential phosphorylation sites (Ser/Thr) at the carboxyl tail of the receptor in order to delineate the receptor phosphorylation sites and the agonist-induced desensitization and internalization. The Ser and Thr were substituted to alanine, and the corresponding mutants were transiently and stably expressed in HEK293 cells. We found that only two residues, i.e. Thr(358) and Ser(363), were phosphorylated, with Ser(363) being critical for the DPDPE-induced phosphorylation of the receptor. Furthermore, using alanine and aspartic acid substitutions, we found that the phosphorylation of the receptor is hierarchical, with Ser(363) as the primary phosphorylation site. Here, we demonstrated that DPDPE-induced rapid receptor desensitization, as measured by adenylyl cyclase activity, and receptor internalization are intimately related to phosphorylation of Thr(358) and Ser(363), with Thr(358) being involved in the receptor internalization.

Sequential phosphorylation of protein band 3 by Syk and Lyn tyrosine kinases in intact human erythrocytes: identification of primary and secondary phosphorylation sites

AbstractTreatment of intact human erythrocytes with pervanadate induces Tyr (Y)-phosphorylation of the transmembrane protein band 3; in parallel, the activity of the immunoprecipitated tyrosine kinases Syk and Lyn is increased. When erythrocytes are incubated with pervanadate together with PP1, a specific inhibitor of Src kinases, including Lyn, the Y-phosphorylation of band 3 is only partially reduced. Indeed, the PP1-resistant phosphorylation of band 3 precedes and is a prerequisite for its coimmunoprecipitation with Lyn, which interacts with the phosphoprotein via the SH2 domain of the enzyme, as proven by binding competition experiments. Upon recruitment to primarily phosphorylated band 3, Lyn catalyzes the secondary phosphorylation of the transmembrane protein. These data are consistent with the view that band 3 is phosphorylated in intact erythrocytes by both PP1-resistant (most likely Syk) and PP1-inhibited (most likely Lyn) tyrosine kinases according to a sequential phosphorylation process. Similar radiolabeled peptide maps are obtained by tryptic digestion of32P-band 3 isolated from either pervanadate-treated erythrocytes or red cell membranes incubated with exogenous Syk and Lyn. It has also been demonstrated by means of mass spectrometry that the primary phosphorylation of band 3 occurs at Y8 and Y21, while the secondary phosphorylation affects Y359 and Y904.

Sequential phosphorylation of protein band 3 by Syk and Lyn tyrosine kinases in intact human erythrocytes: identification of primary and secondary phosphorylation sites

Treatment of intact human erythrocytes with pervanadate induces Tyr (Y)-phosphorylation of the transmembrane protein band 3; in parallel, the activity of the immunoprecipitated tyrosine kinases Syk and Lyn is increased. When erythrocytes are incubated with pervanadate together with PP1, a specific inhibitor of Src kinases, including Lyn, the Y-phosphorylation of band 3 is only partially reduced. Indeed, the PP1-resistant phosphorylation of band 3 precedes and is a prerequisite for its coimmunoprecipitation with Lyn, which interacts with the phosphoprotein via the SH2 domain of the enzyme, as proven by binding competition experiments. Upon recruitment to primarily phosphorylated band 3, Lyn catalyzes the secondary phosphorylation of the transmembrane protein. These data are consistent with the view that band 3 is phosphorylated in intact erythrocytes by both PP1-resistant (most likely Syk) and PP1-inhibited (most likely Lyn) tyrosine kinases according to a sequential phosphorylation process. Similar radiolabeled peptide maps are obtained by tryptic digestion of32P-band 3 isolated from either pervanadate-treated erythrocytes or red cell membranes incubated with exogenous Syk and Lyn. It has also been demonstrated by means of mass spectrometry that the primary phosphorylation of band 3 occurs at Y8 and Y21, while the secondary phosphorylation affects Y359 and Y904.

Identification of the Novel K15 Gene at the Rightmost End of the Kaposi's Sarcoma-Associated Herpesvirus Genome

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a distinct open reading frame called K15 at a position equivalent to the gene encoding LMP2A of Epstein-Barr virus (EBV). K15 isolates from body cavity-based lymphoma (BCBL) cells exhibited a dramatic sequence variation and a complex splicing pattern. However, all K15 alleles are organized similarly with the potential SH2 and SH3 binding motifs in their cytoplasmic regions. Northern blot analysis showed that K15 was weakly expressed in latently infected BCBL-1 cells, and the level of its expression was significantly induced by tetradecanoyl phorbol acetate stimulation. K15 encoded 40- to 55-kDa proteins, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and was localized at the cytoplasm and plasma membrane. To demonstrate the signal-transducing activity of the K15 protein, we constructed a chimeric protein in which the cytoplasmic tail of the human CD8alpha polypeptide was replaced with that of KSHV K15. While the CD8-K15 chimera was not capable of eliciting cellular signal transduction upon stimulation with an anti-CD8 antibody, it significantly inhibited B-cell receptor signaling, as evidenced by a suppression of tyrosine phosphorylation and intracellular calcium mobilization. This inhibition required the putative SH2 or SH3 binding motif in the cytoplasmic region of K15. Biochemical study of CD8-K15 chimeras showed that the cytoplasmic region of K15 was constitutively tyrosine phosphorylated and that the tyrosine residue within the putative SH2 binding motif of K15 was a primary site of phosphorylation. These results demonstrate that KSHV K15 resembles LMP2A in genomic location, splicing pattern, and protein structure and by the presence of functional signal-transducing motifs in the cytoplasmic region. Thus, KSHV K15 is likely a distant evolutionary relative of EBV LMP2A.

Soybean Nodule Sucrose Synthase (Nodulin-100): Further Analysis of Its Phosphorylation Using Recombinant and Authentic Root-Nodule Enzymes

Sucrose synthase (SS) is a known phosphoserine-containing enzyme in legume root nodules and various other plant “sink” tissues. In order to begin to investigate the possible physiological significance of this posttranslational modification, we have cloned a full-length soybean nodule SS (nodulin-100) cDNA and overexpressed it in Escherichia coli. Authentic nodule SS and recombinant wild-type and mutant forms of the enzyme were purified and characterized. We document that a conserved serine near the N-terminus (Ser11) is the primary phosphorylation site for a nodule Ca2+-dependent protein kinase (CDPK) in vitro. Related tryptic digestion and mass spectral analyses indicated that this target residue was also phosphorylated in planta in authentic nodulin-100. In addition, a secondary phosphorylation site(s) in recombinant nodule SS was implicated given that all active mutant enzyme forms (S11A, S11D, S11C, and N-terminal truncation between Ala2 and Arg13) were phosphorylated, albeit weakly, by the CDPK. This secondary site(s) likely resides between Glu14 and Met193 as evidenced by CNBr cleavage and phosphopeptide mapping. Phosphorylation of the recombinant and authentic nodule Ser11 enzymes in vitro by the nodule CDPK had no major effect on the sucrose-cleavage activity and/or kinetic properties. However, phosphorylation decreased the apparent surface hydrophobicity of the recombinant wild-type enzyme, suggesting that this covalent modification could potentially play some role in the documented partitioning of nodulin-100 between the nodule symbiosome/plasma membranes and cytosol in planta.

Agonist-induced, G Protein-dependent and -independent Down-regulation of the μ Opioid Receptor: THE RECEPTOR IS A DIRECT SUBSTRATE FOR PROTEIN-TYROSINE KINASE

The mu opioid receptor (MOR) has been shown to desensitize after 1 h of exposure to the opioid peptide, [D-Ala(2), N-MePhe(4), Gly-ol(5)]enkephalin (DAMGO), largely by the loss of receptors from the cell surface and receptor down-regulation. We have previously shown that the Thr(394) in the carboxyl tail is essential for agonist-induced early desensitization, presumably by serving as a primary phosphorylation site for G protein-coupled receptor kinase. Using a T394A mutant receptor, we determined that Thr(394) was also responsible for mu opioid receptor down-regulation. The T394A mutant receptor displayed 50% reduction of receptor down-regulation (14.8%) compared with wild type receptor (34%) upon 1 h of exposure to DAMGO. Agonist-induced T394A receptor down-regulation was unaffected by pertussis toxin treatment, indicating involvement of a mechanism independent of G protein function. Interestingly, pertussis toxin-insensitive T394A receptor down-regulation was completely inhibited by a tyrosine kinase inhibitor, genistein. Tyrosine kinase inhibition blocked wild type MOR down-regulation by 50%, and the genistein-resistant wild type MOR down-regulation was completely pertussis toxin-sensitive. Following DAMGO stimulation, MOR was shown to be phosphorylated at tyrosine residue(s), indicating that the receptor was a direct substrate for tyrosine kinase action. Mutagenesis of the four intracellular tyrosine residues resulted in complete inhibition of the G protein-insensitive MOR internalization. Therefore, agonist-induced MOR down-regulation appears to be mediated by two distinct cellular signal transduction pathways. One is G protein-dependent and GRK-dependent, which can be abolished by pertussis toxin treatment of wild type MOR or by mutagenesis of Thr(394). The other novel pathway is G protein-independent but tyrosine kinase-dependent, blocked by genistein treatment, and one in which Thr(394) has no regulatory role but phosphorylation of tyrosine residues appears essential.

G Protein-coupled Receptor Kinase 6A Phosphorylates the Na+/H+ Exchanger Regulatory Factor via a PDZ Domain-mediated Interaction

The Na(+)/H(+) exchanger regulatory factor (NHERF) is constitutively phosphorylated in cells, but the site(s) of this phosphorylation and the kinase(s) responsible for it have not been identified. We show here that the primary site of constitutive NHERF phosphorylation in human embryonic kidney 293 (HEK-293) cells is Ser(289), and that the stoichiometry of phosphorylation is near 1 mol/mol. NHERF contains two PDZ domains that recognize the sequence S/T-X-L at the carboxyl terminus of target proteins, and thus we examined the possibility that kinases containing this motif might associate with and phosphorylate NHERF. Overlay experiments and co-immunoprecipitation studies revealed that NHERF binds with high affinity to a splice variant of the G protein-coupled receptor kinase 6, GRK6A, which terminates in the motif T-R-L. NHERF does not associate with GRK6B or GRK6C, alternatively spliced variants that differ from GRK6A at their extreme carboxyl termini. GRK6A phosphorylates NHERF efficiently on Ser(289) in vitro, whereas GRK6B, GRK6C, and GRK2 do not. Furthermore, the endogenous "NHERF kinase" activity in HEK-293 cell lysates is sensitive to treatments that alter the activity of GRK6A. These data suggest that GRK6A phosphorylates NHERF via a PDZ domain-mediated interaction and that GRK6A is the kinase in HEK-293 cells responsible for the constitutive phosphorylation of NHERF.

Human parainfluenza virus type 1 phosphoprotein is constitutively phosphorylated at Ser-120 and Ser-184.

RNA-dependent RNA polymerases of single-stranded, negative-sense RNA viruses comprise a phosphoprotein (P) and a large protein. The constitutive phosphorylation of the P protein in these viruses is highly conserved, yet the functional significance of phosphorylation is enigmatic. To approach this problem, phosphorylation sites were determined in two closely related paramyxovirus P proteins. Sendai virus (SV) is a prototypic paramyxovirus. Previously, using a phosphopeptide mapping technique, the primary constitutive phosphorylation site of SV P protein was mapped to Ser-249. Phosphorylation at Ser-249 is dependent on the presence of Pro-250. Human parainfluenza virus type 1 (HPIV-1) P protein has 66% similarity to SV P protein and its predicted secondary structure is highly similar to that of SV P protein. However, there is no obvious conserved phosphorylation site in HPIV-1 P protein. Using the phosphopeptide mapping strategy, the constitutive phosphorylation sites of HPIV-1 P protein were mapped. The HPIV-1 P protein is primarily phosphorylated at Ser-120. Phosphorylation at Ser-120 is dependent on the presence of Pro-121. It also has a minor phosphorylation site at Ser-184. The sequence at Ser-184 does not match any consensus phosphorylation target site for the known kinases. Significantly, the P proteins from both viruses are constitutively and primarily phosphorylated at one serine and the phosphorylation of that serine is dependent on the presence of a proline on its carboxyl side.

Complete amino acid sequences and phosphorylation sites, determined by Edman degradation and mass spectrometry, of rat parotid destrin- and cofilin-like proteins

β-Adrenergic or cholinergic stimulation of the rat parotid gland was earlier shown to induce dephosphorylation of endogenous destrin- and cofilin-like proteins, which are phosphorylated in resting cells at Ser residues probably present near the N-terminals. The primary structures and phosphorylation sites were determined here. The rat destrin-like protein had a sequence 95% identical to the cDNA-derived sequence of porcine destrin. The rat cofilin-like protein was 98% identical to that of porcine cofilin. Each protein lacked the initiator Met and began with an acetylalanine residue followed by a Ser residue. The N-terminal peptides generated with endoproteinase Asp-N were isolated; they were each phosphorylated at Ser-2. Earlier work had shown that partial cleavage of the phosphorylated destrin- and cofilin-like proteins with cyanogen bromide provides unphosphorylated 16.7- and 18.3-kDa fragments, respectively. It was here confirmed that they contained all the Ser residues other than those present in the N-terminal peptides. From these observations, it was now concluded that the destrin- and cofilin-like proteins are rat parotid destrin and cofilin (non-muscle type), respectively, and that each protein is phosphorylated exclusively at Ser-2 in resting cells and dephosphorylated at this site in response to β-adrenergic or cholinergic stimulation.

A mutation in the Bordetella bronchiseptica bvgS gene results in reduced virulence and increased resistance to starvation, and identifies a new class of Bvg-regulated antigens.

The Bordetella BvgAS signal-transduction system has traditionally been viewed as mediating a transition between two distinct phenotypic phases: the Bvg+ phase, characterized by the expression of adhesins and toxins, and the Bvg-phase, characterized by motility in Bordetella bronchiseptica and by the expression of vrg loci in Bordetella pertussis. In B. bronchiseptica, the Bvg+ phase is necessary and sufficient for respiratory tract colonization whereas the Bvg phase is required for growth under nutrient-limiting conditions. This report describes the characterization of a mutant that is locked in a Bvg-intermediate (Bvg[i]) phase. The mutation conferring this phenotype, designated bvgS-I1, results in a threonine-to-methionine substitution near the primary site of phosphorylation in BvgS. Compared to its Bvg+-phase-locked parent, the Bvg(i) mutant displays increased resistance to nutrient limitation and reduced virulence. Molecular analyses indicate that the mutant has lost the ability to express a subset of Bvg+-phase factors and has gained the ability to express factors unique to the Bvg(i) phase. Although identified by mutation, this work indicates that the Bvg(i) phase is expressed by wild-type B. bronchiseptica in response to certain (semi-modulating) environmental conditions. The identification of Bvg(i)-specific antigens suggests the existence of a new class of Bvg-regulated genes. We hypothesize that BvgAS is capable of mediating the expression of a spectrum of phenotypic phases in response to the various environments encountered as Bordetella travels within and between mammalian hosts.

Identification of Protein Kinase C Phosphorylation Sites on Bovine Rhodopsin

The protein kinase C phosphorylation sites on bovine rhodopsin were identified using proteolytic, phosphoamino acid, mass spectrometric, and peptide sequencing analyses. Tryptic removal of the 9 carboxyl-terminal residues of rhodopsin revealed that a major fraction of the phosphates incorporated by protein kinase C are in a region containing Ser334, Thr335, and Thr336. Phosphoamino acid analysis of the tryptic product established that Ser334 accounts for approximately 65% of the phosphorylation in this region. Analysis of the endoproteinase Asp-N-generated carboxyl terminus of rhodopsin by mass spectrometry and peptide sequencing revealed that Ser338 is also a primary phosphorylation site, with minor phosphorylation of Ser343. Quantitation of high pressure liquid chromatography-separated phosphopeptides, taken together with phosphoamino acid analysis of the tryptic product, revealed that Ser334 and Ser338 were phosphorylated equally and each accounted for approximately 35% of the total phosphorylation; Thr335/336 accounted for just under 20% of the phosphorylation, and Ser343 accounted for 10%. Thus, the primary protein kinase C sites are Ser334 and Ser338, with minor phosphorylation of Thr335/336 and Ser343. Ser334 and Ser338 have recently been identified as the primary sites of phosphorylation of rhodopsin in vivo (Ohguro, H., Van Hooser, J. P., Milam, A. H., and Palczewski, K. (1995) J. Biol. Chem. 270, 14259-14262). Of these sites, only Ser338 is a significant substrate for rhodopsin kinase in vitro. Identification of Ser334 as a primary protein kinase C target in vitro is consistent with protein kinase C modulating the phosphorylation of this site in vivo.

Multiple phosphorylated forms of the Saccharomyces cerevisiae Mcm1 protein include an isoform induced in response to high salt concentrations.

The Saccharomyces cerevisiae Mcm1 protein is an essential multifunctional transcription factor which is highly homologous to human serum response factor. Mcm1 protein acts on a large number of distinctly regulated genes: haploid cell-type-specific genes, G2-cell-cycle-regulated genes, pheromone-induced genes, arginine metabolic genes, and genes important for cell wall and cell membrane function. We show here that Mcm1 protein is phosphorylated in vivo. Several (more than eight) isoforms of Mcm1 protein, resolved by isoelectric focusing, are present in vivo; two major phosphorylation sites lie in the N-terminal 17 amino acids immediately adjacent to the conserved MADS box DNA-binding domain. The implications of multiple species of Mcm1, particularly the notion that a unique Mcm1 isoform could be required for regulation of a specific set of Mcm1's target genes, are discussed. We also show here that Mcm1 plays an important role in the response to stress caused by NaCl. G. Yu, R. J. Deschenes, and J. S. Fassler (J. Biol. Chem. 270:8739-8743, 1995) showed that Mcm1 function is affected by mutations in the SLN1 gene, a signal transduction component implicated in the response to osmotic stress. We find that mcm1 mutations can confer either reduced or enhanced survival on high-salt medium; deletion of the N terminus or mutation in the primary phosphorylation site results in impaired growth on high-salt medium. Furthermore, Mcm1 protein is a target of a signal transduction system responsive to osmotic stress: a new isoform of Mcm1 is induced by NaCl or KCl; this result establishes that Mcm1 itself is regulated.

Phosphorylation of native and truncated isoforms of protein tau by the double-stranded DNA-dependent protein kinase (DNA-PK) shows that the primary phosphorylation sites are localized between amino acid residues 212-231 of the longest tau.

Alzheimer's disease (AD) is pathologically characterized by the appearance of neurofibrillary tangles (NFT), senile plaques, and loss of subpopulation of neuronal cells. The NFT is composed of paired helical filaments (PHT) with extensively modified protein T as its primary constituent. Previously we had reported on the hyperphosphorylation of T by a double-stranded-DNA-stimulated protein kinase (DNA-PK). In this communication, we have compared the DNA-PK mediated phosphorylation of native and truncated TS with that catalyzed by the cAMP-dependent protein kinase (PKA). In addition, we have attempted to map the primary site(s) of phosphorylation of T by DNA-PK. Our results suggest that DNA-PK phosphorylates T at sites substantially different from those targeted by PKA. Furthermore, we show that the primary phosphorylation sites lie between amino acid residues 212-231 (using numbering system for the longest T, which is the isoform with four "repeats" and a 58 amino acid "insert" at the carboxyl- and amino-termini, respectively, of the protein molecule).