Influence of myristoylation, phosphorylation, and deamidation on the structural behavior of the N-terminus of the catalytic subunit of cAMP-dependent protein kinase.

A number of isoenzymes of the catalytic subunit of cAMP-dependent protein kinase arise through posttranslational modifications of the enzyme outside the catalytic domain; the biological significance of these is not yet fully clear. A clustering of sites for such modification exists at the N-terminus of the protein, where myristoylation (of Gly1), phosphorylation (at Ser10), and deamidation of Asn2 have been observed. As the first two are known to govern membrane binding and thus subcellular compartmentalization in some proteins, it was of interest to see whether the local structure of the N-terminus was being influenced by one or more of these modifications. A series of synthetic peptides mimicing the 16 N-terminal residues of the catalytic subunit Calpha was produced covering the full range of possible modifications, singly and in combination, and tested for possible effects on local structure by measuring the circular dichroism under varying polarity. It was found that myristoylation and phosphorylation modify the structure in this region in opposite ways and in a manner designed to amplify the action of a potential myristoyl/electrostatic switch. To what extent deamidation of Asn2 may oppose a potential membrane binding is unknown. Deamidation, however, had no effect on the structure of the peptide either alone or in combination with acylation and/or phosphorylation, suggesting that the change of the nuclear/cytoplasmic disribution in cells caused by deamidation [Pepperkok et al. (2000) J. Cell Biol. 148, 715-726] is due to a more complex signaling mechanism. The structural implications of the data are discussed.

The protein kinase A catalytic subunit Cbeta2: molecular characterization and distribution of the splice variant.

Cbeta2, a 46 kDa splice variant of the Cbeta isoform, is the largest isoform so far described for catalytic subunits from cAMP-dependent protein kinase in mammals. It differs from Cbeta in the first 15 N-terminal residues which are replaced with a 62-residue domain with no similarity to other known proteins. The Cbeta2 protein was identified in cardiac tissue by MS, microsequencing and C-subunit-isoform-selective antibodies. The Cbeta2 protein has a very low abundance of about 2% of total affinity-purified C subunits from bovine cardiac tissue. This, and the similarity of its biochemical properties to Calpha and Cbeta, are probably some of the reasons why the Cbeta2 protein has escaped detection so far. The abundance of the Cbeta2 protein differs dramatically between tissues, with most protein detected in heart, liver and spleen, and the lowest level in testis. Cbeta2 protein shows kinase activity against synthetic substrates, and is inhibited by the protein kinase inhibitor peptide PKI(5-24). The degree of Cbeta2 removal from tissue extracts by binding to PKI(5-24) depends on the cAMP level, i.e. on the dissociation state of the holoenzyme. Two sites in the protein are phosphorylated: Thr-244 in the activation segment and Ser-385 close to the C-terminus. By affinity purification and immunodetection Cbeta2 was found in cattle, pig, rat, mouse and turkey tissue and in HeLa cells. In the cAMP-insensitive CHO 10260 cell line, which has normal Cbeta but is depleted of Calpha, stable transfection with Cbeta2 restored most of the cAMP-induced morphological changes. Cbeta2 is a ubiquitously expressed protein with characteristic properties of a cAMP-dependent protein kinase catalytic subunit.

Intracellular distribution of mammalian protein kinase A catalytic subunit altered by conserved Asn2 deamidation.

The catalytic (C) subunit of protein kinase A functions both in the cytoplasm and the nucleus. A major charge variant representing about one third of the enzyme in striated muscle results from deamidation in vivo of the Asn2 residue at the conserved NH(2)-terminal sequence myrGly-Asn-Ala (Jedrzejewski, P.T., A. Girod, A. Tholey, N. König, S. Thullner, V. Kinzel, and D. Bossemeyer. 1998. Protein Sci. 7:457-469). Because of the increase of electronegativity by generation of Asp2, it is reminiscent of a myristoyl-electrostatic switch. To compare the intracellular distribution of the enzymes, both forms of porcine or bovine heart enzyme were microinjected into the cytoplasm of mouse NIH 3T3 cells after conjugation with fluorescein, rhodamine, or in unlabeled form. The nuclear/cytoplasmic fluorescence ratio (N/C) was analyzed in the presence of cAMP (in the case of unlabeled enzyme by antibodies). Under all circumstances, the N/C ratio obtained with the encoded Asn2 form was significantly higher than that with the deamidated, Asp2 form; i.e., the Asn2 form reached a larger nuclear concentration than the Asp2 form. Comparable data were obtained with a human cell line. The differential intracellular distribution of both enzyme forms is also reflected by functional data. It correlates with the degree of phosphorylation of the key serine in CREB family transcription factors in the nucleus. Microinjection of myristoylated recombinant bovine Calpha and the Asn2 deletion mutant of it yielded N/C ratios in the same range as encoded native enzymes. Thus, Asn2 seems to serve as a potential site for modulating electronegativity. The data indicate that the NH(2)-terminal domain of the PKA C-subunit contributes to the intracellular distribution of free enzyme, which can be altered by site-specific in vivo deamidation. The model character for other signaling proteins starting with myrGly-Asn is discussed.

Analysis of isoaspartate in peptides by electrospray tandem mass spectrometry.

In view of the significance of Asn deamidation and Asp isomerization to isoAsp at certain sites for protein aging and turnover, it was desirable to challenge the extreme analytical power of electrospray tandem mass spectrometry (ESI-MS/MS) for the possibility of a site-specific detection of this posttranslational modification. For this purpose, synthetic L-Asp/L-isoAsp containing oligopeptide pairs were investigated by ESI-MS/MS and low-energy collision-induced dissociation (CID). Replacement of L-Asp by L-isoAsp resulted in the same kind of shifts for all 15 peptide pairs investigated: (1) the b/y intensity ratio of complementary b and y ions generated by cleavage of the (L-Asp/L-isoAsp)-X bond and of the X-(L-Asp/L-isoAsp) bond was decreased, and (2) the Asp immonium ion abundance at m/z 88 was also decreased. It is proposed that the isoAsp structure hampers the accepted mechanism of b-ion formation on both its N- and C-terminal side. The b/y ion intensity ratio and the relative immonium ion intensity vary considerably, depending on the peptide sequence, but the corresponding values are reproducible when recorded on the same instrument under identical instrumental settings. Thus, once the reference product ion spectra have been documented for a pair of synthetic peptides containing either L-Asp or L-isoAsp, these identify one or the other form. Characterization and relative quantification of L-Asp/L-isoAsp peptide mixtures are also possible as demonstrated for two sequences for which isoAsp formation has been described, namely myrG-D/isoD-AAAAK (deamidated peptide 1-7 of protein kinase A catalytic subunit) and VQ-D/isoD-GLR (deamidated peptide 41-46 of human procollagen alpha 1). Thus, the analytical procedures described may be helpful for the identification of suspected Asn deamidation and Asp isomerization sites in proteolytic digests of proteins.

The amino terminus of PKA catalytic subunit--a site for introduction of posttranslational heterogeneities by deamidation: D-Asp2 and D-isoAsp2 containing isozymes.

Conserved deamidation of PKA catalytic subunit isozymes Calpha and Cbeta--more than 25% at Asn2 in vivo in both cases--has been shown to yield Asp2- and isoAsp2-containing isozymes (Jedrzejewski PT, Girod A, Tholey A, König N, Thullner S, Kinzel V, Bossemeyer D, 1998, Protein Sci 7:457-469). Isoaspartate formation in proteins in vivo is indicative of succinimide intermediates involved in both the initial deamidation reaction as well as the "repair" of isoAsp to Asp by the action of protein L-isoaspartyl (D-aspartyl) O-methyl transferase (PIMT). L-Succinimide is prone to racemization to D-succinimide, which may hydrolyze to D-isoAsp- and D-Asp-containing diastereomers with, respectively, no and poor substrate character for PIMT. To analyze native PKA catalytic subunit from cardiac muscle for these isomers the N-terminal tryptic peptides (T1) of the enzyme were analyzed following procedures refined specifically with a set of corresponding synthetic peptides. The methods combined high resolution high-performance liquid chromatography and a new mass spectrometric procedure for the discrimination between Asp- and isoAsp-residues in peptides (Lehmann et al., 2000). The results demonstrate the occurrence of D-isoAsp- and D-Asp-containing T1 fragments in addition to the L-isomers. The small amount of the L-isoAsp isomer, representing only part of the D-isoAsp isomer, and the relatively large amounts of the L-Asp and D-Asp isomers argues for an effective action of PIMT present in cardiac tissue.

The human papillomavirus type 16 E5 protein modulates phospholipase C-gamma-1 activity and phosphatidyl inositol turnover in mouse fibroblasts.

The human papillomavirus type 16 E5 (HPV16-E5) protein is a membrane protein that has been associated with malignant growth. The protein affects growth factor-mediated signal transduction in a ligand-dependent manner. We show now that E5 expression in A31 fibroblasts results in an increased level of diacylglycerol (DAG) and inositol phosphates. Immunoprecipitation of phospholipase C-gamma-1 (PLC-gamma-1) with specific antibodies and immunoblotting with anti-phosphotyrosine antibodies reveal a large increase in tyrosine phosphorylation of the enzyme in E5-expressing cells compared to control vector-transfected cells. This activation of tyrosine phosphorylation is growth factor independent. In addition, an enhanced formation of phosphatidic acid (PA) was observed in E5 cells. This increase did not result from activation of phospholipase D (PLD), although the enzyme was activatable by treatment with phorbol ester Thus, a phosphohydrolase-mediated DAG synthesis from PLD-produced PA can be excluded. The observed effects were not further enhanced by EGF showing that the presence of the growth factor is not necessary for maintaining permanent activation of PLC-gamma-1 in E5-expressing cells. The DAG- and inositol phosphate-mediated signal cascade within the cells is thus effectively uncoupled from external control via EGF and its receptor in the presence of E5 protein.

Clostridium botulinum C2 toxin delays entry into mitosis and activation of p34cdc2 kinase and cdc25-C phosphatase in HeLa cells.

The Clostridium botulinum C2 toxin ADP-ribosylates monomeric actin, thereby inducing disassembly of actin filaments, alteration of focal adhesions, and rounding of cells. After treatment with C2 toxin, cells stop to proliferate but remain viable for about 2 days. In view of reported correlations between the structure of the actin cytoskeleton and cell cycle transition, the effects of C2 toxin on the G(2)/M phase transition of the cell division cycle were studied. Since C2 toxin delayed entry into mitosis in HeLa cells, those enzymes which control entry into mitosis, the cyclin-dependent protein kinase mitosis-promoting factor (MPF) and the phosphatase cdc25-C were examined after treatment of synchronized cells with C2 toxin. MPF is composed of the regulatory cyclin B and the enzymatic p34cdc2 kinase subunits. For its activation at the G2/M border, p34cdc2 needs to be associated with cyclin B and additionally dephosphorylated at Tyr-15 by the specific phosphatase cdc25-C. Treatment of synchronized cells in S or G2 phase with C. botulinum C2 toxin prevented p34cdc2 protein kinase activation by inhibiting its tyrosine dephosphorylation at the G2/M border. Furthermore, the activity of cdc25-C phosphatase was decreased after treatment of cells with C2 toxin. Our results suggest that the prevented activation of the mitotic inducers p34cdc2 kinase and cdc25-C phosphatase represents the final downstream events in the action of C2 toxin resulting in a G(2) phase cell cycle delay in synchronized HeLa cells.

Direct effects of phosphorylation on the preferred backbone conformation of peptides: a nuclear magnetic resonance study.

Control of protein activity by phosphorylation appears to work principally by inducing conformational change, but the mechanisms so far reported are dependent on the structural context in which phosphorylation occurs. As the activity of many small peptides is also regulated by phosphorylation, we decided to investigate possible direct consequences of this on the preferred backbone conformation. We have performed 1H nuclear magnetic resonance (NMR) experiments with short model peptides of the pattern Gly-Ser-Xaa-Ser, where Xaa represents Ser, Thr, or Tyr in either phosphorylated or unphosphorylated form and with either free or blocked amino and carboxy termini. The chemical shifts of amide protons and the 3JNH-Halpha coupling constants were estimated from one-dimensional and two-dimensional scalar correlated spectroscopy (COSY) spectra at different pH values. The results clearly indicate a direct structural effect of serine and threonine phosphorylation on the preferred backbone dihedrals independent of the presence of charged groups in the surrounding sequence. Tyrosine phosphorylation does not induce such a charge-independent effect. Additionally, experiments with p-fluoro- and p-nitro-phenylalanine-containing peptides showed that the mere presence of an electronegative group on the aromatic ring of tyrosine does not produce direct structural effects. In the case of serine and threonine phosphorylation a strong dependence of the conformational shift on the protonation level of the phosphoryl group could be observed, showing that phosphorylation induces the strongest effect in its dianionic, i.e., physiological, form. The data reveal a hitherto unknown mechanism that may be added to the repertoire of conformational control of peptides and proteins by phosphorylation.

A conserved deamidation site at Asn 2 in the catalytic subunit of mammalian cAMP-dependent protein kinase detected by capillary LC-MS and tandem mass spectrometry.

The N-terminal sequence myr-Gly-Asn is conserved among the myristoylated cAPK (protein kinase A) catalytic subunit isozymes Calpha, Cbeta, and Cgamma. By capillary LC-MS and tandem MS, we show that, in approximately one third of the Calpha and Cbeta enzyme populations from cattle, pig, rabbit, and rat striated muscle, Asn 2 is deamidated to Asp 2. This deamidation accounts for the major isoelectric variants of the cAPK C-subunits formerly called CA and CB. Deamidation also includes characteristic isoaspartate isomeric peptides from Calpha and Cbeta. Asn 2 deamidation does not occur during C-subunit preparation and is absent in recombinant myristoylated Calpha (rCalpha) from Escherichia coli. Deamidation appears to be the exclusive pathway for introduction of an acidic residue adjacent to the myristoylated N-terminal glycine, verified by the myristoylation negative phenotype of an rCalpha(Asn 2 Asp) mutant. This is the first report thus far of a naturally occurring myr-Gly-Asp sequence. Asp 2 seems to be required for the well-characterized (auto)phosphorylation of the native enzyme at Ser 10. Our results suggest that the myristoylated N terminus of cAPK is a conserved site for deamidation in vivo. Comparable myr-Gly-Asn sequences are found in several signaling proteins. This may be especially significant in view of the recent knowledge that negative charges close to myristic acid in some proteins contribute to regulating their cellular localization.

Structure of the LAV6 peptide: a nucleation site for the correct receptor-induced refolding of the CD4-binding domain of HIV1 gp 120.

LAV44 and LAV15 (lymphadenopathy-associated virus) peptides of the CD4-binding region of gp 120 per se bind to the CD4 receptor (Reed and Kinzel, Biochemistry 30: 4521-4528, 1991; Lasky et al., Cell 50:975-985, 1987). Depending on the environment, the LAV peptides exhibit the ability to switch cooperatively between beta-sheet and helical conformation when solvent polarity is changed past a critical point. This property, which is dependent on the amino acid sequence LPCR, is crucial for receptor binding (Reed and Kinzel, Proc. Natl. Acad. Sci. U.S.A. 90:6761-6765, 1993). Structure determination with 2D-NMR-spectroscopy reveals that LAV6 peptide (sequence: TLPCRI) has a well-defined structure, partially exhibiting inverse gamma-turn conformation in aqueous solution. Quantitative evaluation of the NMR data discloses 90% trans-conformation for the peptide bond between leucine and proline. The psi- and phi-angles fall into the typical range for amino acids located in turns. On the other hand, the amino acid sequence C-terminal to the LPCR tetrad has been shown to fold atypically in the absence of these residues. All these results show that the short sequence of LAV6 peptide, with the central amino acids LPCR, displays a matrix-independent structure and may, therefore, act as a conformational template for forming secondary structure in the intact CD4-binding domain of gp 120.