Familienrecht

Protein kinase C (PKC) consists of a family of closely related subtypes which differ in their localization and activation properties. Our previous studies have suggested a role for PKC in the regulation of noradrenaline (NA) release from the human neuroblastoma SH-SY5Y. Here we have used two approaches to characterize the PKC subtypes present in SH-SY5Y cells. Firstly, the PCR was used to show that SH-SY5Y cells contain mRNA encoding PKC subtypes alpha, beta, gamma, delta, epsilon and zeta. Secondly, immunoblotting showed that SH-SY5Y cells express PKC subtypes alpha, epsilon and zeta at the protein level. Prolonged (48 h) exposure of cells to the phorbol ester phorbol 12-myristate 13-acetate (PMA; 100 nM) resulted in a marked decrease in the amounts of PKC-alpha and PKC-epsilon, with no change in levels of PKC-zeta. Prolonged PMA treatment had no significant effect on K(+)-evoked NA release from SH-SY5Y cells, whereas carbachol-evoked release was increased 2.2-fold. However, prolonged exposure to PMA completely inhibited the ability of acute (12 min) PMA treatment to enhance both K(+)- and carbachol-evoked NA release. The specific PKC inhibitor RO 31-7459 (10 microM) was found to inhibit K(+)- and carbachol-evoked release by 27% and 68% respectively. RO 31-7549 also completely inhibited the ability of acute PMA treatment to enhance release. These data suggest that PKC-alpha and/or PKC-epsilon play an essential role in the regulation of PMA-enhanced K(+)- and carbachol-evoked NA release in SH-SY5Y cells.

Section: Discussionsupporting

confidence: 92%

Section: Detection Of Mrnas Encoding Pkc Subtypes By Using the Pcrmentioning

confidence: 99%

A role for protein kinase C subtypes α and ε in phorbol-ester-enhanced K+- and carbachol-evoked noradrenaline release from the human neuroblastoma SH-SY5Y

Turner

Rumsby

Walker

et al. 1994

“…Note that: (1) all PKC isoforms migrated with molecular-mass standards of 80-90 kDa (the slightly higher apparent molecular masses of some PKC isoforms observed here may reflect the fact that samples in Laemmli buffer were not heated); (2) 80-90 kDa PKC-C in rat brain cytosol was a double with a third lower band at approx. 60 kDa (these three bands have also been noted by others [6]), whereas rat adipocyte PKC-C was essentially a single 85 kDa band, with a faint highermolecular-mass band observed only in some blots; and (3) only one PKC-y band was absorption-sensitive when assayed in the presence of excess peptide antigen. Immunoreactive PKC-,8, PKC-y, PKC-e and PKC-C were detectable in both cytosol and membrane fractions of control and insulin-treated adipocytes (Figs.…”

Section: Resultssupporting

confidence: 70%

Effects of insulin and phorbol esters on subcellular distribution of protein kinase C isoforms in rat adipocytes

Farese

Standaert

François

et al. 1992

Effects of insulin and phorbol esters on subcellular distribution of protein kinase C (PKC) isoforms were examined in rat adipocytes. Both agonists provoked rapid decreases in cytosolic, and/or increases in membrane, immunoreactive PKC-alpha, PKC-beta, PKC-gamma, and PKC-epsilon. Effects of phorbol esters on PKC-alpha redistribution to the plasma membrane, however, were much greater than those of insulin. In contrast, insulin, but not phorbol esters, stimulated the translocation of PKC-beta to the plasma membrane, and provoked changes in PKC-zeta redistribution. Neither agonist altered subcellular distribution of PKC-delta, which was detected only in membrane fractions. Our findings indicate that insulin and phorbol esters have overlapping and distinctly different effects on the subcellular redistribution of specific PKC isoforms.

“…Peptide-KLH conjugates were used tor rabbit injection and antibody production. PKC isoenzyme sequences were obtained trom Ono et al (1988Ono et al ( , 1989, Makowske et al (1988) andHenrich (1991). Peptides were synthesized on the basis ot sequences unique to the a, /7, Ry, 6, e or 5 isotorms ot PKC or a sequence conserved in all six torms ('con-pep') (see the Results section).…”

Section: Pdepjmentioning

confidence: 99%

“…PCs ROSPKC+PDE Figure 7 Phosphorylation of POE by ROS PKC in the presence of different peptides (Table 1). Peptides were synthesized based on sequences unique to the a, /7, y, 6, e or C isoforms of PKC or to a sequence conserved in all six forms (Ono et al, 1988(Ono et al, , 1989Makowske et al, 1988;Henrich, 1991). Peptides corresponded to sequences from the V3 region of PKCoc, PKCf) and PKCy, or to sequences from C-terminus (V5 region) of PKCU, PKCe and PKCC.…”

Section: Pdepjmentioning

confidence: 99%

Phosphorylation of bovine rod photoreceptor cyclic GMP phosphodiesterase

Udovichenko

Cunnick

Gonzales

et al. 1993

The cyclic GMP phosphodiesterase (PDE) of retinal rods plays a key role in phototransduction and consists of two catalytic subunits (PDE alpha and PDE beta) and two identical inhibitory subunits (PDE gamma). Here we report that PDE alpha and PDE gamma are phosphorylated by protein kinase(s) C (PKC) from brain and rod outer segments (ROS). These same two types of PKC also phosphorylate PDE alpha in trypsin-activated PDE (without PDE gamma). In contrast, cyclic-AMP-dependent protein kinase catalytic subunit phosphorylates both PDE alpha and PDE beta, but not PDE gamma. This kinase does not phosphorylate trypsin-activated PDE. The synthetic peptides AKVISNLLGPREAAV (PDE alpha 30-44) and KQRQTRQFKSKPPKK (PDE gamma 31-45) inhibited phosphorylation of PDE by PKC from ROS. These data suggest that sites (at least one for each subunit) for phosphorylation of PDE by PKC are localized in these corresponding regions of PDE alpha and PDE gamma. Isoenzyme-specific PKC antibodies against peptides unique to the alpha, beta, gamma, delta, epsilon and zeta isoforms of protein kinase C were used to show that a major form of PKC in ROS is PKC alpha. However, other minor forms were also present.