phosphothreonine and staurosporine-aglycone

The phosphorylation and dephosphorylation of cytoskeletal proteins regulate the shape of eukaryotic cells. To elucidate the role of serine/threonine protein phosphatases (PP) in this process, we studied the effects of calyculin A (CLA), a potent and specific inhibitor of protein phosphatases 1 (PP-1) and 2A (PP-2A) on the cytoskeletal structure of cultured human umbilical vein endothelial cells (HUVECs). The addition of CLA (5 min) caused marked alterations in cell morphology, such as cell constriction and bleb formation. Microtubules and F-actin were reorganized, becoming markedly condensed around the nucleus. Although the fluorescence intensity of phosphoamino acids was not significantly different according to immunocytochemistry between cells with and without CLA, polypeptides of 135, 140, 158, and 175 kDa were specifically phosphorylated on serine and/or threonine residues. There was no significant effect on tyrosine residues. The effects of CLA on cytoskeletal changes and protein phosphorylation were almost completely inhibited by the non-selective kinase inhibitor, K-252a. The effect of CLA on cell morphology was at least 100 times more potent than that of okadaic acid, consistent with the inhibitory potency against PP-1. The catalytic subunit of PP-1 was also identified in HUVECs by Western blotting with its monoclonal antibody antibody. These results suggest that PP-1 is closely involved in sustaining the normal structure of the cytoskeleton.

Topics: Actins; Animals; Blotting, Western; Carbazoles; Cell Nucleus; Cells, Cultured; Cytoskeleton; Endothelium, Vascular; Enzyme Inhibitors; Humans; Indole Alkaloids; Marine Toxins; Mice; Microtubules; Oxazoles; Phosphoprotein Phosphatases; Phosphorylation; Phosphoserine; Phosphothreonine; Protein Kinase Inhibitors; Protein Phosphatase 1; Umbilical Veins

Using an in situ kinase assay we have identified kinases that are elevated in some multidrug resistant cells. Kinases were detected by measurement of 32P incorporation in proteins that were renatured after being subjected to SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes [Ferrell and Martin: J Biol Chem 264:20723-20729, 1989; Mol Cell Biol 10:3020-3026, 1990]. Kinases at 79, 84, and 92 kDa showed increased activity in the multidrug resistant human KB-V1 cells as compared to the sensitive parental KB-3-1 cells. The KB-V1 multidrug resistant cell line exhibited a 170 kDa membrane associated kinase activity that was not present in the parental drug sensitive line. The 170 kDa kinase activity was not affected by Ca++, phosphatidylserine, or cAMP, but was diminished after incubation in the presence of the kinase inhibitors staurosporine, K252a and KT5720. The 170 kDa kinase activity phosphorylated mainly threonine, with no evidence of tyrosine phosphorylation, and was not identical to either the multidrug resistance associated P-glycoprotein or the EGF receptor. Other multidrug resistant cell lines also showed elevated 170 kDa kinase activity, such as the human breast cancer MCF-7/Adr(R) and murine melanoma B16/Adr(R) cells, but the activity was not present in murine leukemia P-388 sensitive or multidrug resistant cells.

Topics: Alkaloids; Animals; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B, Member 1; Breast Neoplasms; Calcium; Carbazoles; Carrier Proteins; Clone Cells; Cyclic AMP; Drug Resistance; Electrophoresis, Polyacrylamide Gel; Humans; Indole Alkaloids; Indoles; KB Cells; Melanoma, Experimental; Membrane Glycoproteins; Mice; Neoplasm Proteins; Phosphatidylserines; Phosphorylation; Phosphothreonine; Phosphotyrosine; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Pyrroles; Staurosporine; Transfection; Tumor Cells, Cultured; Tyrosine