calyculin-a and decamethrin

The liver is a central organ involved in many aspects of physiology and disease. Signaling properties of hepatocytes, the main liver cell type, are of special interest in metabolic diseases and in regeneration. For this reason we investigated the phosphoproteome of the mouse liver cell line Hepa1-6 by stable isotope labeling by amino acids in cell culture (SILAC) and high resolution MS. Using stringent statistical evaluation criteria, we obtained 5433 phosphorylation sites on 1808 proteins. The phosphoproteome encompasses all major protein classes, including a large number of transcription factors. We compared control and phosphatase inhibitor treated cells by SILAC. This enabled ready identification of in vivo phosphorylation sites by sequencing the more abundant, inhibitor induced version of the peptide while still observing the endogenous version. We employed a mixture of pervanadate for blocking protein tyrosine phosphatases (PTPs) and calyculin A and deltamethrin for blocking the activities of serine/threonine phosphatases. Interestingly, these commonly used inhibitors in standard concentrations affected only 28% of the phosphopeptides by at least two-fold. The unaffected sites may be substrates of phosphatases that are not efficiently inhibited, have slow kinetic or sites that are almost stoichiometric in normally growing cells. Finally, we devised a triple labeling strategy comprising control cells, stimulated cells, and phosphatase treated cells to derive an upper bound on phosphorylation occupancy.

Topics: Animals; Cell Line, Tumor; Chromatography, Liquid; Computational Biology; Hepatocytes; Insulin; Liver; Marine Toxins; Mice; Nitriles; Oxazoles; Phosphoprotein Phosphatases; Phosphoproteins; Phosphorylation; Protein Tyrosine Phosphatases; Proteome; Pyrethrins; Substrate Specificity; Tandem Mass Spectrometry; Transcription Factors; Vanadates

Thrombin-induced Ca2+ mobilization, activation of Ca2+/calmodulin-dependent myosin light chain (MLC) kinase (MLCK), and increased phosphorylation of MLCs precede and are critical to endothelial cell (EC) barrier dysfunction. Net MLC dephosphorylation after thrombin is nearly complete by 60 min and involves type 1 phosphatase (PPase 1) activity. We now report that thrombin does not alter total PPase 1 activity in EC homogenates but rather decreases myosin-associated PPase 1 activity. The PPase 1 inhibitor calyculin fails to prevent thrombin-induced MLC dephosphorylation. However, thrombin significantly increased the activity of Ca2+-dependent PPase 2B in EC homogenates (approximately 1.5- to 2-fold), with PPase 2B activation correlating with phosphorylation of the PPase 2B catalytic subunit. Western immunoblotting revealed PPase 2B to be present in cytoskeletal EC fractions, with specific PPase 2B inhibitors such as cyclosporin (200 nM) and deltamethrin (100 nM to 1 microM) attenuating thrombin-induced cytoskeletal protein dephosphorylation, including EC MLC dephosphorylation. These results suggest a model whereby thrombin-inducible contraction is determined by the phosphorylation status of EC MLC regulated by the balance between EC MLCK, PPase 1 (constitutive), and PPase 2B (inducible) activities.

Topics: Animals; Calcium; Cattle; Cells, Cultured; Endothelium, Vascular; Enzyme Inhibitors; Ionomycin; Kinetics; Marine Toxins; Myosin Light Chains; Myosin-Light-Chain Kinase; Nitriles; Oxazoles; Phosphoprotein Phosphatases; Phosphorylation; Pulmonary Artery; Pyrethrins; Thrombin