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Query: UMLS:C0848332 (Spots)
 453 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Collagen fibrils suppressed serum- or epidermal growth factor (EGF)-inducible DNA synthesis of human fibroblasts. The phosphorylation of cellular proteins upon these mitogenic stimulation was analyzed by two-dimensional polyacrylamide gel electrophoresis in order to reveal a possible interference of collagen fibrils with the cellular mitogenic signal transduction pathway coupled with the protein phosphorylation-dephosphorylation reaction. Spots of phosphorylated proteins numbered 192 on plain plastic which were reduced to 143 on collagen fibrils. More than half of them were matched between the two substrates, most of which were much more weakly phosphorylated on collagen fibrils. EGF stimulated the phosphorylation of these proteins of cells on plastic. Among them a protein with an approximate molecular weight of 27K and an isoelectric point of 5.3 was early and highly responsive to EGF, phosphorylation of which seemed to be catalyzed mainly by protein kinase C and tyrosine kinase. Collagen fibrils significantly suppressed this phosphorylation. The present study demonstrates that collagen fibrils modulate the growth-associated protein phosphorylation of cells, which seems to lead to the suppression of DNA synthesis.
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PMID:Suppression of growth-associated phosphorylation of proteins of fibroblasts by collagen fibrils. 880 90

We report a novel method to identify protein kinase C (PKC) substrates. Tissue lysates were fractionated by ion exchange chromatography and used as substrates in in vitro kinase reactions. The phosphorylated proteins were separated using two-dimensional gel electrophoresis. Spots that contained isolated phosphoproteins were excised and digested with trypsin. The tryptic peptides were analyzed using mass spectrometry. While several of the proteins identified using this technique represent known PKC substrates, we identified a new PKC substrate in the initial screen. This protein, sm22, is expressed in smooth muscle cells and served well as a substrate for PKC in vitro. Sm22 is predominantly associated with the actin cytoskeleton. Upon activation of PKC in vivo, sm22 dissociates from the actin cytoskeleton and is distributed diffusely in the cytoplasm. Our data strongly suggest that phosphorylation by PKC controls the intracellular localization of sm22. This demonstrates that our approach, using a complex mixture of proteins as in vitro kinase substrates and subsequently identifying the newly phosphorylated proteins by mass spectrometry, is a powerful method to identify new kinase substrates.
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PMID:Identification of the smooth muscle-specific protein, sm22, as a novel protein kinase C substrate using two-dimensional gel electrophoresis and mass spectrometry. 1093 58

Using a proteomics approach, we examined the post-translational changes in cytosolic proteins when isolated Malpighian tubules of Aedes aegypti were stimulated for 1 min with the diuretic peptide aedeskinin-III (AK-III, 10(-7) mol l(-1)). The cytosols of control (C) and aedeskinin-treated (T) tubules were extracted from several thousand Malpighian tubules, subjected to 2-D electrophoresis and stained for total proteins and phosphoproteins. The comparison of C and T gels was performed by gel image analysis for the change of normalized spot volumes. Spots with volumes equal to or exceeding C/T ratios of +/-1.5 were robotically picked for in-gel digestion with trypsin and submitted for protein identification by nanoLC/MS/MS analysis. Identified proteins covered a wide range of biological activity. As kinin peptides are known to rapidly stimulate transepithelial secretion of electrolytes and water by Malpighian tubules, we focused on those proteins that might mediate the increase in transepithelial secretion. We found that AK-III reduces the cytosolic presence of subunits A and B of the V-type H(+) ATPase, endoplasmin, calreticulin, annexin, type II regulatory subunit of protein kinase A (PKA) and rab GDP dissociation inhibitor and increases the cytosolic presence of adducin, actin, Ca(2+)-binding protein regucalcin/SMP30 and actin-depolymerizing factor. Supporting the putative role of PKA in the AK-III-induced activation of the V-type H(+) ATPase is the effect of H89, an inhibitor of PKA, on fluid secretion. H89 reverses the stimulatory effect of AK-III on transepithelial fluid secretion in isolated Malpighian tubules. However, AK-III does not raise intracellular levels of cAMP, the usual activator of PKA, suggesting a cAMP-independent activation of PKA that removes subunits A and B from the cytoplasm in the assembly and activation of the V-type H(+) ATPase. Alternatively, protein kinase C could also mediate the activation of the proton pump. Ca(2+) remains the primary intracellular messenger of the aedeskinins that signals the remodeling of the paracellular complex apparently through protein kinase C, thereby increasing transepithelial anion secretion. The effects of AK-III on active transcellular and passive paracellular transport are additive, if not synergistic, to bring about the rapid diuresis.
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PMID:Signaling to the apical membrane and to the paracellular pathway: changes in the cytosolic proteome of Aedes Malpighian tubules. 1915 Dec 7