Novel isoforms of CaBP 33/37 (annexin V) from mammalian brain: structural and phosphorylation differences that suggest distinct biological roles.

Two calcium-dependent phospholipid- and membrane-binding proteins have been purified from bovine brain. These are termed CaBP33 and CaBP37. Complete sequence analysis has revealed that these two proteins are isoforms of annexin V. Despite an apparent difference of 4 kDa between the two proteins on SDS-PAGE, only two amino-acid substitutions were found. These are, in CaBP33, Ser-36 and Lys-125 and in CaBP37, Thr-36 and Glu-125. This corresponds to a mass difference of 15 Da. This was confirmed by electrospray mass spectrometric analysis. Both isoforms can be phosphorylated substoichiometrically in vitro by protein kinase C at residue Thr-22.

Multisite phosphorylation of the 80 kDa (MARCKS) protein kinase C substrate in C3H/10T1/2 fibroblasts. Quantitative analysis of individual sites by solid-phase microsequencing.

A synthetic peptide, KKKKRFSFKKSFKLSGFSFKK, containing the phosphorylation sites of the acidic 80-87 kDa protein kinase C substrate was used to identify phosphopeptides in enzyme digests of this protein from mouse fibroblast C3H/10T1/2 cells. Stimulation of phosphorylation occurred, in vivo, with TPA at Ser7, Ser11 and Ser18, and, with two less potent phorbol esters, at Ser7 and Ser18. Okadaic acid effected a net phosphorylation of Ser7 and/or Ser11. Solid-phase sequencing showed that, in vitro, the order of initial rate of phosphorylation was Ser11 greater than Ser7 greater than Ser18, while Ser18 was preferentially phosphorylated when either Ser7 or Ser11 was occupied. No significant phosphorylation of Ser15 was detected.

Purification of a 20 kDa phosphoprotein from epithelial cells and identification as a myosin light chain. Phosphorylation induced by enteropathogenic Escherichia coli and phorbol ester.

Previous studies on the mechanism of enteropathogenic Escherichia coli (EPEC) infection have revealed an increase in the phosphorylation state of a number of proteins in human laryngeal HEp-2 cells. The most prominent was an acidic phosphoprotein(s) of Mr 20-21 kDa. The present study reports: (a) a simple method for purification of phosphorylated 20 kDa protein; (b) identification of the 20 kDa phosphoprotein as myosin light chain; and (c) that the phorbol ester, TPA, also increased the phosphorylation of the 20 kDa myosin light chain. In contrast to the effects of EPEC, TPA stimulation resulted in the dissociation of myosin from the cytoskeleton to the cytosol.

Programmed cell death in sympathetic neurons: a study by two-dimensional polyacrylamide gel electrophoresis using computer image analysis.

The technique of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) coupled with computer image analysis was used in this study to examine changes in protein expression occurring during the onset of programmed cell death (PCD) in rat sympathetic neurons following withdrawal of nerve growth factor (NGF). Sympathetic neurons from superior cervical ganglia of postnatal day-one Wistar rats were cultured in the presence of NGF for 24 h and then either maintained in the presence of NGF or deprived of NGF for a period of 8 h. To label the proteins being synthesised, neurons were cultured in the presence of L-[35S]methionine for a further 2 h under the same conditions but with 3% of the normal methionine concentration. Neuronal proteins were then analysed by 2-D PAGE using immobilised pH gradient (IPG) gel strips in the first dimension. For the second dimension a custom-built electrophoresis system capable of running multiple sodium dodecyl sulfate (SDS)-PAGE slab gels in a vertical configuration, with good temperature control (+/- 0.7 degrees C) was used and is described in this paper. Proteins resolved on the dried gels were visualised using storage phosphor technology and the digitised images subjected to rigorous analysis using the QUEST II software system. Seventeen proteins whose relative synthesis decreased and four proteins that increased upon NGF withdrawal were located and are documented.

Multiple isoforms of a protein kinase C inhibitor (KCIP-1/14-3-3) from sheep brain. Amino acid sequence of phosphorylated forms.

A potent inhibitor of protein kinase C (PKC), inhibitor protein-1 (KCIP-1), isolated from sheep brain has been shown to consist of eight isoforms by reverse-phase HPLC. Direct protein sequence analysis has revealed these to be the same as those of 14-3-3 protein, described as an activator of tyrosine and tryptophan hydroxylases involved in neurotransmitter biosynthesis. The N-termini of KCIP-1 isoforms were shown to be acetylated, and secondary structure predictions revealed a high degree of alpha-helix with an amphipathic nature. KCIP-1 showed no inhibitory activity towards protein kinase M (the catalytic fragment of PKC) and had no effect on the activities of three other protein kinases, cAMP-dependent protein kinase, Ca2+/calmodulin-dependent protein kinase II and casein kinase 2. Four forms of KCIP-1 were shown to be substrates for PKC in vitro, but none were phosphorylated by the other protein kinases mentioned above.

Proteomic comparison of human and great ape blood plasma reveals conserved glycosylation and differences in thyroid hormone metabolism.

Most blood plasma proteins are glycosylated. These glycoproteins typically carry sialic acid-bearing sugar chains, which can modify the observed molecular weights and isoelectric points of those proteins during electrophoretic analyses. To explore changes in protein expression and glycosylation that occurred during great ape and human evolution, we subjected multiple blood plasma samples from all these species to high-resolution proteomic analysis. We found very few species-specific differences, indicating a remarkable degree of conservation of plasma protein expression and glycosylation during approximately 12 million years of evolution. A few lineage-specific differences in protein migration were noted among the great apes. The only obvious differences between humans and all great apes were an apparent decrease in transthyretin (prealbumin) and a change in haptoglobin isoforms (the latter was predictable from prior genetic studies). Quantitative studies of transthyretin in samples of blood plasma (synthesized primarily by the liver) and of cerebrospinal fluid (synthesized locally by the choroid plexus of the brain) confirmed approximately 2-fold higher levels in chimpanzees compared to humans. Since transthyretin binds thyroid hormones, we next compared plasma thyroid hormone parameters between humans and chimpanzees. The results indicate significant differences in the status of thyroid hormone metabolism, which represent the first known endocrine difference between these species. Notably, thyroid hormones are known to play major roles in the development, differentiation, and metabolism of many organs and tissues, including the brain and the cranium. Also, transthyretin is known to be the major carrier of thyroid hormone in the cerebrospinal fluid, likely regulating delivery of this hormone to the brain. A potential secondary difference in retinoid (vitamin A) metabolism is also noted. The implications of these findings for explaining unique features of human evolution are discussed.

Structure and sites of phosphorylation of 14-3-3 protein: role in coordinating signal transduction pathways.

The 14-3-3 family are homo- and heterodimeric proteins whose biological role has been unclear for some time, although they are now gaining acceptance as a novel type of 'adaptor' protein that modulates interactions between components of signal transduction pathways, rather than by direct activation or inhibition. It is becoming apparent that phosphorylation of the binding partner and possibly also the 14-3-3 proteins may regulate these interactions. 14-3-3 isoforms interact with a novel phosphoserine (Sp) motif on many proteins, RSX1,2SpXP. The two isoforms that interact with Raf-1 are phosphorylated in vivo on Ser185 in a consensus sequence motif for proline-directed kinases. The crystal structure of 14-3-3 indicates that this phosphorylation could regulate interaction of 14-3-3 with its target proteins. We have now identified a number of additional phosphorylation sites on distinct mammalian and yeast isoforms.

Proteomics: a major new technology for the drug discovery process.

Proteomics is a new enabling technology that is being integrated into the drug discovery process. This will facilitate the systematic analysis of proteins across any biological system or disease, forwarding new targets and information on mode of action, toxicology and surrogate markers. Proteomics is highly complementary to genomic approaches in the drug discovery process and, for the first time, offers scientists the ability to integrate information from the genome, expressed mRNAs, their respective proteins and subcellular localization. It is expected that this will lead to important new insights into disease mechanisms and improved drug discovery strategies to produce novel therapeutics.

Proteomic definition of normal human luminal and myoepithelial breast cells purified from reduction mammoplasties.

Normal human luminal and myoepithelial breast cells separately purified from a set of 10 reduction mammoplasties by using a double antibody magnetic affinity cell sorting and Dynabead immunomagnetic technique were used in two-dimensional gel proteome studies. A total of 43,302 proteins were detected across the 20 samples, and a master image for each cell type comprising a total of 1,738 unique proteins was derived. Differential analysis identified 170 proteins that were elevated 2-fold or more between the two breast cell types, and 51 of these were annotated by tandem mass spectrometry. Muscle-specific enzyme isoforms and contractile intermediate filaments including tropomyosin and smooth muscle (SM22) alpha protein were detected in the myoepithelial cells, and a large number of cytokeratin subclasses and isoforms characteristic of luminal cells were detected in this cell type. A further 134 nondifferentially regulated proteins were also annotated from the two breast cell types, making this the most extensive study to date of the protein expression map of the normal human breast and the basis for future studies of purified breast cancer cells.