Characteristics of thyroid protein kinase C

Abstract

Thyroid protein kinase C (PKc) from cytosols of porcine and rat thyroid glands has been characterized using histone H1 or endogenous proteins as substrates. As in many other tissues histone HI is by far the preferred exogenous substrate of thyroid PKc. Kinetic studies with H1 showed that, compared to rat thyroids, porcine glands are particularly rich in PKc, the predominant kinase activity in this tissue. The CAMP-dependent protein kinase (PKa) level, on the contrary, is very similar in both rat and porcine thyroids. Consequently, for the same type of tissue, there may be great species differences in the PKc level and the ratios between PKc and PKa kinase activities.Chromatographic properties of thyroid PKc are similar to those described in other tissues (one major peak followed by a small shoulder) except that elution of the main peak can vary depending on the nature of the salt gradient ( M 55 mM for NaCl and 15 mM for sodium phosphate). In the first case PKc is completely separated from the PKa activity, in the second it is coeluted with the peak of PKa type I.The one-dimensional PAGE pattern of proteins phosphorylated by porcine PKc is very similar to the pattern obtained by rat enzyme. Protein bands of 18 kDa, 22-25 kDa and 32-36 kDa are specific substrates of the thyroid PKc, after in vitro phosphorylation of cytosol proteins.A great difference in Ca2+ requirement for PKc activation was noted, depending whether histone HI or endogenous proteins were substrates. As in other tissues, calcium was absolutely necessary for phosphorylation of histone H1 by PKc. The addition of calcium was not absolutely necessary when endogenous proteins were the substrates, either for the activation of the enzyme or for phosphorylation of the PKc-specific substrates. Almost the same rate of phosphorylation was obtained with or without calcium in the incubation medium. However the one-dimensional PAGE pattern of phosphorylated proteins was different in the presence or absence of calcium. While addition of calcium was not absolutely necessary for the phosphorylation of a great number of proteins by the PKc, its presence was indispensable for the phosphorylation of certain endogenous substrates. However, calcium alone, in the absence of phospholipids had no effect on the phosphorylation of these proteins.Endogenous proteins, phosphorylated by the PKc only when calcium was present, were resolved by the twodimensional PAGE into several distinct spots with molecular masses of 32 -35 kDa and PI range of 5 -7.5.Our results suggest that only very low concentrations of Ca2+, if any, are sufficient for the phosphorylation of many endogenous substrates. Higher Ca2 + concentrations are necessary for the phosphorylation of specific proteins. It could be that modulation of Ca2+ concentration plays a role in the selection of protein substrates phosphorylated by PKc.

Increased Steady-State Levels of CUGBP1 in Myotonic Dystrophy 1 Are Due to PKC-Mediated Hyperphosphorylation

“…We also demonstrated that recombinant PKCα and βII directly phosphorylate His-CUGBP1 in an in vitro kinase reaction. Several PKC isozymes translocate to the nucleus upon activation and regulate nuclear events such as transcription, splicing and mRNA stability via phosphorylation of nuclear targets such as lamin B and histone H1 (Fields et al, 1988;Martelli et al, 2006;Martelli et al, 2003;Omri et al, 1987). It is also possible that CUGBP1 is phosphorylated in the cytoplasm and is efficiently translocated to the nucleus.…”

Protein kinase C-dependent phosphorylation of annexins I and II in mesangial cells

“…All incubations were carried out at 30°C for 3 min. Total endogenous protein phosphorylation was measured on samples (50 pll) of the reaction mixture spotted on 2.1 cm-diameter filter paper discs (Whatman 3MM) and processed as previously described (Omri et al, 1987). The remaining fraction (250 ,1) was mixed with an equal volume in lysis buffer containing the following; 10 mM Tris/HCl, pH 7.4, 50 mM NaCl, 1 % Nonidet P40, 0.5 % sodium deoxycholate, 5 mM EGTA, 1 mg/ml BSA, 1 mM phenylmethanesulphonyl fluoride, 100 units/ml aprotinin, 1 #sg/ml each of antipain, leupeptin and pepstatin, and 1 mM orthovanadate, 25 mM NaF, 5 mM p-nitrophenyl phosphate and 10 mM sodium pyrophosphate.…”

Aspirin prevention of NMDA‐induced neuronal death by direct protein kinase Cζ inhibition

“…We evaluated PKCζ activity by measuring the incorporation of 32 P into specific substrate in aliquots in which the reaction had been stopped, on 2.1‐cm diameter Whatman filter discs, by dropping the discs into 10% trichloroacetic acid solution supplemented with 0.1 m m ATP as a carrier. Filter discs were decontaminated as described previously (Omri et al . 1987).…”