Possible regulation of epidermal growth factor-receptor tyrosine autophosphorylation by calcium and G proteins in chemically permeabilized rat UMR106 cells

• Published in: Archives of oral biology 1999-02, Vol.44 (2), p.157-171
• Main Authors: Kawase, T, Orikasa, M, Oguro, A, Burns, D.M
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - pharmacology; Animals; Calcium; Calcium - administration & dosage; Calcium - pharmacology; Calcium Signaling; Carcinoma - metabolism; Cell Membrane Permeability; Culture Media; Dentistry; Dose-Response Relationship, Drug; Enzyme Inhibitors - pharmacology; Epidermal growth factor; Epidermal Growth Factor - pharmacology; ErbB Receptors - drug effects; ErbB Receptors - metabolism; G proteins; Genistein - pharmacology; GTP-Binding Proteins - physiology; Humans; Osteoblastic cells; Osteoblasts - metabolism; Osteosarcoma - metabolism; Phosphoproteins - metabolism; Phosphorylation; Protein Tyrosine Phosphatases - antagonists & inhibitors; Protein Tyrosine Phosphatases - drug effects; Protein Tyrosine Phosphatases - metabolism; Protein-Tyrosine Kinases - antagonists & inhibitors; Protein-Tyrosine Kinases - drug effects; Protein-Tyrosine Kinases - metabolism; Rats; Signal Transduction - physiology; Time Factors; Tumor Cells, Cultured; Tyrosine - drug effects; Tyrosine - metabolism; Tyrosine phosphorylation; Vanadates - pharmacology
• ISSN: 0003-9969; 1879-1506
• DOI: 10.1016/S0003-9969(98)00105-8

A model using chemically permeabilized cells was developed to examine mechanisms that regulate protein tyrosine phosphorylation in osteoblastic cells. Using either permeabilized UMR106 osteoblastic or A431 (reference) cells, epidermal growth factor (EGF)-induced cellular tyrosine phosphorylation, and whether there are previously unrecognized interactions between this transduction pathway and Ca 2+- or G protein-dependent signalling pathways, were investigated. Both permeabilized cell types, when maintained in non-supplemented cytoplasmic substitution solution (basic CSS), responded to EGF (1–100 ng/ml) with dose-dependent increases in tyrosine phosphorylation. A complex and time-dependent pattern of phosphotyrosine-containing proteins resulted, but the profile of tyrosine phosphorylated proteins was appreciably less complex than in intact cells. Supplementation of basic CSS with MgATP restored the normal complexity of the profiles for EGF-induced tyrosine phosphorylation proteins in both permeabilized cell lines and produced a more sustained accumulation of phosphoprotein products in A431 cells. Adding Ca 2+ (≤10 −6M), with or without exogenous MgATP, dose-dependently attenuated EGF-induced tyrosine phosphorylation of EGF receptors (EGFR) and other substrates in UMR106 cells, but was less effective in A431 cells. In both cell types, genistein, an inhibitor of tyrosine kinases, was more effective in attenuating EGF-induced receptor tyrosine phosphorylation in permeabilized cells. Similarly, orthovanadate, an inhibitor of protein tyrosine phosphatases, stimulated the accumulation of phosphoprotein products more effectively in permeabilized cells. Thus, the permeabilization preserves many features of intact cells while facilitating manipulation of intracellular conditions. NaF reproducibly produced a significant vanadate-like action in permeabilized cells that was somewhat stronger than its effect on intact cells. In contrast, the well-known inhibition of tyrosine phosphorylation by phorbol 12-myristate 13-acetate (PMA) was less effective in permeabilized cells than in intact cells; these actions of PMA were Ca 2+-dependent. In addition, guanylyl-imidodiphosphate (Gpp(NH)p) attenuated tyrosine phosphorylation in UMR106 cells, and this effect was specifically blocked by guanosine 5′-O-(2-thiodiphosphate) (GDP βs). These results strongly suggest that there is crosstalk between EGFR-activated tyrosine phosphorylation/dephosphorylation pathways and both Ca 2+- an