Involvement of Protein Kinase Cϵ in the Stimulation of Anionic Amino Acid Transport in Cultured Human Fibroblasts

Abstract

Protein kinase C (PKC) activation stimulates transport system X AG ؊ for anionic amino acids in cultured human fibroblasts (Franchi-Gazzola, R., Visigalli, R., Bussolati, O., and Gazzola, G. C. (1994) FEBS Lett. 352, 109 -112). To identify which PKC isoform is responsible for this effect, aspartate transport through system X AG ؊ , PKC activity, and the subcellular distribution of PKC isoforms have been studied before and after treatment with phorbol 12,13-dibutyrate (PDBu) in fibroblasts maintained at low serum for 1 (control cells) or 7 days (quiescent cells). In control cells aspartate transport and PKC activity in the particulate fraction were stimulated by short term PDBu treatment; both stimulatory effects were down-regulated by a prolonged exposure to the phorbol. In contrast, in quiescent cells aspartate transport and particulate PKC activity were higher than control under basal conditions, unaffected by a short term PDBu treatment, and lowered by a prolonged incubation with the phorbol. In both control and quiescent cells a short term PDBu treatment modified PKC␣ distribution, increasing its membrane-associated fraction. PKC␦ was mostly in the soluble fraction and scarcely sensitive to PDBu. A brief exposure to PDBu increased membrane-associated PKC⑀ in control but not in quiescent cells. In these cells ⑀ isoform was found exclusively in the particulate fraction even in PDBuuntreated cells. A prolonged PDBu treatment caused a partial down-regulation of membrane-associated PKC⑀ in control cells and its marked decrease in quiescent cells. It is concluded that PKC-dependent changes in system X AG ؊ activity parallel the behavior of PKC⑀, thus suggesting a specific role for this isoform in system X AG ؊ regulation.

Protein Kinase Cε-dependent Regulation of Cystic Fibrosis Transmembrane Regulator Involves Binding to a Receptor for Activated C Kinase (RACK1) and RACK1 Binding to Na+/H+ Exchange Regulatory Factor

“…7. ⑀V1-2 dose dependently blocked the interaction between rhPKC⑀ and rhRACK1 with an IC 50 aliquot (3.26 M) of rhPKC⑀ (Fig. 7, first lane).…”

“…PtdSer and dioctanylglycerol were required for the binding. An EC 50 (effective concentration) for PKC⑀ binding of 6 M was calculated from Hill plots of binding data.…”

“…Selective activation of PKC⑀ or inhibition of PKC⑀ translocation has specific consequences in other cell types. Secretion of amyloid precursor protein (49), anionic amino acid transport (50), activation of cardiac L-type Ca 2ϩ channels (13), and protection of cardiac myocytes by ischemic preconditioning (51,52) are all regulated by PKC⑀. RACK1 has been implicated in some of these effects of PKC⑀; however, the intracellular signaling mechanism still must be determined.…”

“…8D, RACK1 binds to NHERF1 in a dose-dependent manner. An EC 50 , calculated from the data of Fig. 8D, is 3.1 g of RACK1, equivalent to a nominal concentration of 50.1 mM.…”

Glutamate transporters in platelets: EAAT1 decrease in aging and in Alzheimer’s disease

“…Moreover, reduced expression of GLT1 (EAAT2) in global transient ischemia animal model [56] and in ALS patients [30,47] seems responsible for the elevation of extracellular glutamate concentration and neuronal death. Molecular and/or functional characterizations of glutamate transporters were performed in different cell culture models from CNS and peripheral tissues, in order to investigate the regulation of their expression, translocation and activity by various endogenous and exogenous factors [11][12][13]21,35,38,49].…”