Physiological Aspects of Surface Protein Kinase Activity

Kübler, Dieter; Pyerin, Walter; Fehst, M.; Kinzel, V.

doi:10.1007/978-3-642-70664-6_14

Cited by 10 publications

(4 citation statements)

References 27 publications

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“…Most ofthese regulatory phosphorylation reactions are catalyzed by protein kinases localized in intracellular compartments of the cell. Within the last 10 years, surface-located protein kinases (ecto-protein kinases, PKE) have been detected in various intact cells (Mastro and Rozengurt, 1976;Chiang et al, 1979;Kubler et al, 1982Kubler et al, , 1983Kubler et al, , 1986Kubler et al, , 1989Amano et al, 1984;Sonka et al, 1989). Recently, the presence of ecto-protein kinase activity at the surface of neuronal cells has been demonstrated, and specific phosphorylated substrates of the neuronal ecto-protein kinase have been identified (Ehrlich et al, 1986(Ehrlich et al, , 1990Tsuji et al, 1988;Zhang et al, 1988).…”

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confidence: 99%

Ecto‐Protein Kinase and Surface Protein Phosphorylation in PC12 Cells: Interactions with Nerve Growth Factor

Pawłowska

Hogan

Kornecki

et al. 1993

Journal of Neurochemistry

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Abstract:The phosphorylation of surface proteins by ectoprotein kinase has been proposed to play a role in mechanisms underlying neuronal differentiation and their responsiveness to nerve growth factor (NGF). PC 12 clones represent an optimal model for investigating the mode of action of NGF in a homogeneous cell population. In the present study we obtained evidence that PC12 cells possess ectoprotein kinase and characterized the endogenous phosphorylation of its surface protein substrates. PC12 cells maintained in a chemically defined medium exhibited phosphorylation of proteins by [y-32P]ATP added to the medium at time points preceding the intracellular phosphorylation of proteins in cells labeled with 32P,. This activity WBS abolished by adding apyrase or trypsin to the medium but was not sensitive to addition of an excess of unlabeled P,. As also expected from ecto-protein kinase activity, PC 12 cells catalyzed the phosphorylation of an exogenous protein substrate added to the medium, dephospho-a-casein, and this activity competed with the endogenous phosphorylation for extracellular ATP. Based on these criteria, three protein components migrating in sodium dodecyl sulfate gels with apparent molecular weights of 105K, 39K, and 20K were identified as e.wIusive substrates of ecto-protein kinase in PC12 cells. Of the phosphate incorporated into these proteins from extracellular ATP, 75-87% was found in phosphothreonine. The phosphorylation of the 39K protein by ecto-protein kinase did not require Mg2+, implicating this activity in the previously demonstrated regulation of Ca2+-dependent, high-affinity norepinephrine uptake in PC 12 cells by extracellular ATP. The protein kinase inhibitor K-252a inhibited both intra-and extracellular protein phosphorylation in intact PC 12 cells. Its hydrophilic analogue K-252b, had only minimal effects on intracellular protein phosphorylation but readily inhibited the phosphorylation of specific substrates of ecto-protein kinase in PC12 cells incubated with extracellular ATP, suggesting the involvement of ecto-protein kinase in the reported inhibition of NGF-induced neurite extension by K-252b. Preincubation of PC12 cells with 50 ng/ml of NGF for 5 min stimulated the activity of ecto-protein kinase toward all its endogenous substrates. Exposure of PC12 cells to the same NGF concentration for 3 days revealed another substrate of ecto-protein kinase, a 53K protein, whose surface phosphorylation is expressed only after NGF-induced neuronal differentiation. In the concentration range (10-100 p h f ) at which 6-thioguanine blocked NGF-promoted neurite outgrowth in PC12 cells, 6-thioguanine effectively inhibited the phosphorylation of specific proteins by ecto-protein kinase. This study provides the basis for continued investigation of the involvement of ecto-protein kinase and its surface protein substrates in neuronal differentiation, neuritogenesis, and synaptogenesis. Key Words: Protein phosphorylationEcto-protein kinase-Surface phosphoproteins-ExtracelMar ATP-Nerve growth factor...

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confidence: 99%

Ecto‐Protein Kinase and Surface Protein Phosphorylation in PC12 Cells: Interactions with Nerve Growth Factor

Pawłowska

Hogan

Kornecki

et al. 1993

Journal of Neurochemistry

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“…The specificity for ATP and the selectivity for 3T6 cells indicate, that external ATP interact with specific sites on the cell surface. These sites could be enzymes or receptors (Weisman et al, 1988;Gordon, 1986;Kinzel et al, 1986;Kubler et al, 1986;Burnstock and Kennedy, 1985;Williams, 1987) Exogenous adenine nucleotides serve as substrates for cell surface nucleotidases, which catalyze their hydrolysis to adenosine. Adenosine was found to inhibit the growth of several cell lines (Henderson and Scott, 1981;Fox and Kelley, 1978;Ishii and Green, 19731, but the proliferation of 3T6 cells is not inhibited upon addition of adenosine, up to 1 mM (Weisman et al, 1988).…”

Section: Discussionmentioning

confidence: 99%

ATP‐resistant variants of transformed mouse fibroblasts

Belzer

Friedberg

1989

Journal Cellular Physiology

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Addition of ATP to cultures of transformed mouse fibroblasts, 3T6 cells, resulted in cell growth inhibition, whereas the growth of the non-transformed counterparts, 3T3 cells, was only slightly affected. The inhibition was found to be specific for adenine nucleotides, and concentration dependent. At relatively low concentrations (e.g., 1.0 mM) the effect of ATP was cytostatic, whereas at higher concentrations (e.g., 1.0 mM) a cytotoxic effect was exerted. ATP-resistant variants of 3T6 cells were selected by exposure of cultures to gradually elevated concentrations of ATP. The variants were found to resemble the non-transformed counterparts, 3T3 cells, more than the 3T6 parent cells, by the following criteria: ATP-induced alterations in the membrane potential, changes in membrane permeability, cell growth inhibition, and colony formation on soft agar. The data indicate that long exposure of the transformed cells to external ATP results in redifferentiation and reduction in their tumorigenicity.

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“…Protein kinases utilizing extracellular ATP have been described on several cell types, including neuronal cell lines and primary neurons, and have been linked to several biological events, such as cell-cell interaction, communication, adhesion, phosphorylation of exogenous ligands, reception, and transduction of extracellular signals (Mastro and Rozengurt, 1976;Chiang et al, 1979;Kübler et al, 1982Kübler et al, , 1983Amano et al, 1984;Ehrlich et al, 1986;Kübler et al, 1986;Ehrlich, 1987;Tsuji et al, 1988;Zhang et al, 1988;Kübler et al, 1989;Sonka et al, 1989).…”

Section: Introductionmentioning

confidence: 97%