Characterisation of the N'1 isoform of the cyclic AMP-dependent protein kinase (PK-A) catalytic subunit in the nematode, Caenorhabditis elegans.

Multiple isoforms of the cyclic AMP-dependent protein kinase (PK-A) catalytic (C) subunit, arise as a consequence of the use of alternative splicing strategies during transcription of the kin-1 gene in the nematode, Caenorhabditis elegans. N-myristoylation is a common co-translational modification of mammalian PK-A C-subunits; however, the major isoform (N'3), originally characterised in C. elegans, is not N-myristoylated. Here, we show that N'1 isoforms are targets for N-myristoylation in C. elegans. We have demonstrated the in vivo incorporation of radioactivity into N'1 C-subunit isoforms, following incubation of nematodes with [(3)H]-myristic acid. HPLC and MALDI-TOF MS analysis of proteolytic digests of immunoprecipitates confirmed the presence of myristoyl-glycine in the C-subunit. In order to better understand the impact of the N'1 N-terminal sequence, and its myristoylation, on C-subunit activity, a chimerical C-subunit, consisting of the N'1 N-terminus from C. elegans and a murine core and C-terminal sequence was expressed. Myristoylation had no appreciable effect on the catalytic properties of the chimeric protein. However, the myristoylated chimeric protein did exhibit enhanced apolar targeting compared to the myristoylated wild-type murine polypeptide. This behaviour may reflect the inability of the N'1-encoded N-terminus sequence to correctly dock with a hydrophobic domain on the surface of the C-subunit.

An E. coli over-expression system for multiply-phosphorylated proteins and its use in a study of calcium phosphate sequestration by novel recombinant phosphopeptides.

Phosphoproteins and phosphopeptides were expressed by E. coli to give yields of 30-200mg of purified protein per litre with an average degree of phosphorylation at multiple sites of 61-83%. The method employed two compatible cohabiting plasmids having low and high copy number, expressing a protein kinase and, more abundantly, the substrate (poly)peptide, respectively. It was used to phosphorylate recombinant beta-casein or osteopontin at multiple casein kinase-2 sites. Two constructs were designed to produce shorter peptides containing one or more clusters of phosphorylation sites resembling the phosphate centres of caseins. In the first, a 53-residue 6-His tagged phosphopeptide was expressed at a 5-fold higher molar yield. The second had multiple tandem repeats of a tryptic phosphopeptide sequence to give a similar increase in efficiency. Each recombinant phosphopeptide was purified (30-100mg) and small-angle X-ray scattering measurements showed that they, like certain casein and osteopontin phosphopeptides, sequester amorphous calcium phosphate to form calcium phosphate nanoclusters. In principle, the method can provide novel phosphopeptides for the control of biocalcification or be adapted for use with other kinases and cognate proteins or peptides to study the effect of specific phosphorylations on protein structure. Moreover, the insertion of a phosphate centre sequence, possibly with a linker peptide, may allow thermodynamically stable, biocompatible nanoparticles to be made from virtually any sequence.

Role of calcium phosphate nanoclusters in the control of calcification.

Calcium phosphate nanoclusters are equilibrium particles of defined chemical composition in which a core of amorphous calcium phosphate is sequestered within a shell of casein phosphopeptides. Sequence analyses and a structure prediction method were applied to secreted phosphoproteins of known importance in controlling calcification, and eight noncasein phosphoproteins were identified as containing one or more subsequences capable of forming nanoclusters. Small-angle X-ray scattering was used to confirm that a plasmin phosphopeptide of one of the identified proteins, osteopontin, formed a novel type of calcium phosphate nanocluster in which the radius of the amorphous calcium phosphate core was four times larger than is typical of casein nanoclusters. A thermodynamic treatment of nanocluster formation identified the factors of importance in determining the equilibrium size of the core, and showed how a nanocluster solution could be thermodynamically stable yet supersaturated with respect to the mineral phase of bones and teeth. It is suggested that the ability of some secreted phosphoproteins to form nanoclusters is physiologically important for the control or inhibition of calcification in soft and mineralized tissues, the extracellular matrix and a wide range of biofluids, including milk and blood.

siRNA-mediated knockdown of a splice variant of the PK-A catalytic subunit gene causes adult-onset paralysis in C. elegans.

In C. elegans, the PK-A catalytic subunit is encoded by kin-1, which has six 5' exons (N'1-N'6), any one of which may be alternatively spliced onto exon-2. Here we describe a novel siRNA-based strategy to knockdown the expression levels of the N'3 and N'4 splice variants. We show that this technique can effectively knockdown expression of the targeted isoforms without affecting expression of the other kin-1 splice variants. We suggest that this strategy could be widely used in C. elegans to investigate the function of genes with alternative first exons. Moreover, we report a novel role for the N'3 kin-1 variant. Whereas knockdown of the N'4 variant results in no obvious phenotype, loss of the N'3 variant leads to paralysis and an egg-laying defect in the adult, suggesting a deficit in the function of the neuromuscular junction. The function of the N'3 variant is discussed in relation to the known function of PK-A in regulation of the release of neurotransmitters from many presynaptic termini.

Molecular characterisation of cAMP-dependent protein kinase (PK-A) catalytic subunit isoforms in the male tick, Amblyomma hebraeum.

The cAMP-dependent protein kinase (protein kinase A, PK-A) plays a central role in the regulation of diverse aspects of cellular activity. Specifically, PK-A appears to play a key controlling role in the maturation of spermatids. Using a PCR-based approach, with degenerate primers from the highly conserved regions of the PK-A catalytic (C) subunit in combination with 5' and 3' RACE, we have cloned three cDNAs for the PK-A C-subunit of the male tick, Amblyomma hebraeum. The three cDNAs have open reading frames of 1059, 1275 and 1404bp which encode proteins of 40.6, 48.2 and 52.5kDa, respectively. These transcripts appear to arise from 5' alternative splicing of RNA derived from a single gene for the PK-A C-subunit. One isoform (AH-PK-A C1), in common with PK-A C-subunits from a range of species, contains a consensus sequence for N-myristoylation. RT-PCR and Western blot experiments suggest that the three splice variants are expressed ubiquitously; however, expression of the myristoylatable AH-PK-A C1 isoform is predominant in all investigated tissues (accessory gland, midgut, Malpighian tubules, salivary gland, testis and immature spermatids). There is no evidence for a sperm-specific PK-A C-subunit (Cs) in tick sperm; however, tyrosine protein phosphorylation, previously shown to be modulated by PK-A activity during mammalian sperm maturation, was observed in tick sperm.

Expression of multiple isoforms of the cAMP-dependent protein kinase (PK-A) catalytic subunit in the nematode, Caenorhabditis elegans.

The cAMP-dependent protein kinase (protein kinase A, PK-A) plays a central role in the regulation of many aspects of eukaryotic cellular activity. In the free-living nematode, Caenorhabditis elegans, two genes encode PK-A-like catalytic subunits. The kin-1 gene has the potential to generate, through alternative splicing events, a multiplicity of catalytic subunit isoforms; in contrast, the F47F2.1b gene appears to encode just a single authentic catalytic subunit-like protein. Here, we report on the occurrence of, and developmental changes in the expression of, polypeptide products of these genes in both C. elegans and the closely related nematode, C. briggsae. Polypeptides derived from the F47F2.1 gene and its orthologue were detected in mixed stage populations of C. elegans and C. briggsae, respectively. Likewise, a number of polypeptides arising as a result of alternative splicing of transcripts from kin-1, or its orthologue in C. briggsae, were identified in mixed stage populations of nematodes. These isoforms included polypeptides with N-termini encoded by exons N'1 or N'4 and C-termini encoded by exons 7 or N. The expression of isoforms with an N-terminus encoded by the N'1 exon is of significance because the amino acid sequence encoded by this exon encompasses an N-myristoylation motif. Isoform abundance appears to be related to developmental stage. Substantial differences in polypeptide expression profiles can be seen in embryonic and adult nematodes. The functional significance of this PK-A catalytic subunit isoform diversity is discussed.

Cumulative mutagenesis of the basic residues in the 201-218 region of insulin-like growth factor (IGF)-binding protein-5 results in progressive loss of both IGF-I binding and inhibition of IGF-I biological action.

We have reported previously that mutation of two conserved nonbasic amino acids (G203 and Q209) within the highly basic 201-218 region in the C-terminal domain of IGF-binding protein-5 (IGFBP-5) decreases binding to IGFs. This study reveals that cumulative mutagenesis of the 10 basic residues in this region, to create the C-Term series of mutants, ultimately results in a 15-fold decrease in the affinity for IGF-I and a major loss in heparin binding. We examined the ability of mutants to inhibit IGF-mediated survival of MCF-7 cells and were able to demonstrate that this depended not only upon the affinity for IGF-I, but also the kinetics of this interaction, because IGFBP-5 mutants with similar affinity constants (K(D)) values, but with different association (Ka) and dissociation (Kd) rate values, had markedly different inhibitory properties. In contrast, the affinity for IGF-I provided no predictive value in terms of the ability of these mutants to enhance IGF action when bound to the substratum. Instead, these C-Term mutants appeared to enhance the actions of IGF-I by a combination of increased dissociation of IGF-IGFBP complexes from the substratum, together with dissociation of IGF-I from IGFBP-5 bound to the substratum. These effects of the IGFBPs were dependent upon binding to IGF-I, because a non-IGF binding mutant (N-Term) was unable to inhibit or enhance the actions of IGF-I. These results emphasize the importance of the kinetics of association/dissociation in determining the enhancing or inhibiting effects of IGFBP-5 and demonstrate the ability to generate an IGFBP-5 mutant with exclusively IGF-enhancing activity.

Characterisation of an N-terminal variant of acetyl-CoA carboxylase-alpha: expression in human tissues and evolutionary aspects.

mRNA encoding a variant acetyl-CoA carboxylase (ACC)-alpha isozyme, transcribed from a downstream promoter, PIII, was detected in human tissues. Such exon 5A-containing transcripts (E5A-mRNA) encode ACC-alpha with a distinct N-terminus, with 15/17 residues identical to those encoded by the ovine mRNA. In the current study we used antisera directed against the E5A N-terminus to verify that ovine E5A translates are present in tissues consistent with the distribution of E5A-mRNA. The presence of E5A alters the context of adjacent regulatory phosphorylation sites in E6, which may indicate altered regulation of activity for this isozyme. Sequences with high identity to the proximal promoter of PIII and E5A are present in the mouse and rat ACC-alpha genes, however, the coding region of E5A is not conserved, and E5A transcripts are not detected in tissues. Thus E5A must have been present in a common ancestor of rodents, primates, and ruminants, and has become nonfunctional in the former. A minor human PIII-derived mRNA containing an additional 111-bp sequence encoded by a downstream exon, E5B, was also detected. E5B encodes an in-frame stop-codon such that the E5A open-reading frame is terminated, however, ACC-alpha translation may be re-initiated from a downstream AUG in E6, potentially generating an isozyme lacking the N-terminal phosphorylation sites. Transcription of human ACC-alpha from at least three promoters and the potential to generate ACC-alpha isozymes with differential susceptibilities to phosphorylation indicate that the regulation of fatty acid synthesis in human tissues is likely to be complex.

The inhibition of cAMP-dependent protein kinase by full-length hepatitis C virus NS3/4A complex is due to ATP hydrolysis.

Hepatitis C virus (HCV) is an important cause of chronic liver disease, but the molecular mechanisms of viral pathogenesis remain to be established. The HCV non-structural protein NS3 complexes with NS4A and has three enzymatic activities: a proteinase and a helicase/NTPase. Recently, catalytically inactive NS3 fragments containing an arginine-rich motif have been reported to interact with, and inhibit, the catalytic subunit of cAMP-dependent protein kinase (PKA C-subunit). Here we demonstrate that full-length, catalytically active NS3/4A, purified from recombinant baculovirus-infected insect cells, is also able to inhibit PKA C-subunit in vitro. This inhibition was abrogated by mutation of either the arginine-rich motif or the conserved helicase motif II, both of which also abolished NTPase activity. As PKA C-subunit inhibition was also enhanced by poly(U) (an activator of NS3 NTPase activity), we hypothesized that PKA C-subunit inhibition could be due to NS3/4A-mediated ATP hydrolysis. This was confirmed by experiments in which a constant ATP concentration was maintained by addition of an ATP regeneration system--under these conditions PKA C-subunit inhibition was not observed. Interestingly, the mutations also abrogated the ability of wild-type NS3/4A to inhibit the PKA-regulated transcription factor CREB in transiently transfected hepatoma cells. Our data are thus not consistent with the previously proposed model in which the arginine-rich motif of NS3 was suggested to act as a pseudosubstrate inhibitor of PKA C-subunit. However, in vivo effects of NS3/4A suggest that ATPase activity may play a role in viral pathology in the infected liver.