Rat brain glial cells in primary culture and subculture contain the δ, ϵ and ζ subspecies of protein kinase C as well as the conventional subspecies

Gott, Amanda L.; Mallon, Barbara S.; Paton, Alex; Groome, Nigel P.; Rumsby, Martin G.

doi:10.1016/0304-3940(94)90619-x

Cited by 31 publications

(14 citation statements)

References 19 publications

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“…Blots were repeated at least twice with fresh samples of C6 cells at approximately similar stages of growth. The experiments confirmed [16] that C6 cells express PKC subspecies ~, fl~, fin, Y, 6, ~ and ~" at the expected molecular weights with the exception of the fl~ and flH PKC subspecies which were always detected as bands of 40 kDa. We assume that these are proteolytic breakdown products since these were competed out with the appropriate peptide and appear in 6-week-old rat brain samples together with bands at the correct 80 kDa position.…”

Section: Protein Kinase C Subspecies Protein Expression During C6supporting

confidence: 61%

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Protein expression of the α, γ, δ and ε subspecies of protein kinase C changes as C6 glioma cells become contact inhibited and quiescent in the presence of serum

et al. 1995

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Total protein kinase C (PKC) activity and protein expression of the c~ and 6 subspecies of PKC increases markedly as C6 glioma cells grow from low cell density to the contactinhibited quiescent state (also known as Go) in the presence of serum. At the same time protein expression of PKC subspecies y and e decreases while the iBl, ~ID t and ~ subspecies did not change. Serum deprivation of growing C6 glioma cells does not induce the same changes in PKC subspecies protein expression. The findings support the growing view that there are significant differences between the Go states brought about by contact inhibition or serum deprivation.

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Section: Protein Kinase C Subspecies Protein Expression During C6supporting

confidence: 61%

“…Polyclonal antisera to the C-terminal sequences of rat PKC subspecies ct, ill, fllI, y, 6, e, ( and r/were raised and characterised as described [14,16]. C6 cells were rinsed twice in flasks with cold Tris-saline and were then scraped into homogenisation buffer (HB) without Triton X-100.…”

Section: Immunodetection Of Pkc Subspeciesmentioning

confidence: 99%

Protein expression of the α, γ, δ and ε subspecies of protein kinase C changes as C6 glioma cells become contact inhibited and quiescent in the presence of serum

et al. 1995

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“…Astrocytes in culture express different isoforms of PKC (Gott et al, 1994); however, the most abundant isoform of PKC in astrocytes is PKC-␤II (Slepko et al, 1999). Therefore, it is likely that the activation of this PKC isoform in astrocytes may play a prominent role in PKC-transduced signals.…”

Section: Introductionmentioning

confidence: 98%

Translocation of protein kinase C‐βII in astrocytes requires organized actin cytoskeleton and is not accompanied by synchronous RACK1 relocation

Pascale¹,

Alkon²,

Grimaldi³

2004

Glia

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Protein kinase C (PKC)-betaII is the most abundant PKC isoform in astrocytes. Upon activation, this isoform of PKC translocates from the cytosol to the plasma membrane (PM). In this study, we investigated in astrocytes the modality of PKC-betaII translocation as far as the participation of the receptor for activated C kinase-1 (RACK1) and the requirement for intact cytoskeleton in the process. In astrocytes, Western blots and immunocytochemistry coupled to confocal microscopic quantitative analysis showed that after 5 min of phorbol-12-myristate-13-acetate (PMA) exposure, native PKC-betaII, but not PKC-betaI, is relocated efficiently from the cytosol to the PM. Translocation of PKC-betaII was not associated with synchronous RACK1 relocation. Furthermore, the quantity of PM-associated PKC-betaII that co-immunoprecipitated with PM-bound RACK1 increased following PMA exposure, indicating a post activation binding of the two proteins in the PM. Because RACK1 and PKC-betaII relocation seemed not to be synchronous, we hypothesized that an intermediate interaction with the cytoskeleton was taking place. In fact, we were able to show that pharmacological disruption of actin-based cytoskeleton greatly deranged PKC-betaII translocation to the PM. The requirement for intact actin cytoskeleton was specific, because depolymerization of tubulin had no effect on the ability of the kinase to translocate to the PM. These results indicate that in astrocytes, RACK1 and PKC-betaII synchronous relocation is not essential for relocation of PKC-betaII to the PM. In addition, we show for the first time that the integrity of the actin cytoskeleton plays a specific role in PKC-betaII movements in these cells. We hypothesize that in glial cells, rapidly occurring changes of actin cytoskeleton arrangement may be involved in the fast reprogramming of PKC targeting to specific PM location to phosphorylate substrates in different cellular locations.

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“…The staining for PKC isoforms was performed with in-house polyclonal antibodies against the rat sequences, as detailed elsewhere (Drew et al 1994, Gott et al 1994, Littlebury et al 1997, at dilutions between 1:200 and 1:500 (Eckert et al unpublished). These in-house antibodies were analysed by Western blotting and showed similar protein sizes for PKCbI, bII, g, d, e and z in mouse liver and/or brain lysates, as described previously (Drew et al 1994, Gott et al 1994, Littlebury et al 1997) (data not shown). Preincubation with the respective peptide against which they were raised blocked immunofluorescent staining either partially or completely.…”

Section: Antibodies and Verification Of Staining Patternsmentioning

confidence: 99%

PKC signalling regulates tight junction membrane assembly in the pre-implantation mouse embryo

Eckert

McCallum²,

Mears³

et al. 2004

Reproduction

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Epithelial differentiation including tight junction (TJ) formation occurs exclusively within the trophectoderm (TE) lineage of the mouse blastocyst. Here we examine mechanisms by which TJ protein membrane assembly might be regulated by protein kinase C (PKC) in the embryo. To overcome the inherent staging asynchrony of individual blastomeres within intact embryos, we have used isolated inner cell masses (ICMs) from early blastocysts to induce epithelial differentiation in their outer cells responding to their new cell contact pattern. Two TJ proteins examined retain their order of membrane assembly in isolated ICMs in culture as during normal development (early-assembling ZO-2 and late-assembling ZO-1a 1 ), but this process is highly accelerated. Using six chemical modulators of PKC activity, we show here that PKC signalling is involved in the regulation of TJ membrane assembly. While indolactam-mediated PKC activation stimulates membrane assembly of both TJ proteins, TPAmediated PKC activation stimulates only that of ZO-1a 1 . The PKC inhibitors Ro-31-8220, Ro-31-8425 and Gö 6983 suppress the stimulatory effect of both PKC activators on membrane assembly to varying extents according to inhibitor and TJ protein examined. Gö 6983 similarly inhibits ZO-2 and ZO-1a 1 membrane assembly. PKC inhibition by Gö 6976 appeared to stimulate TJ membrane assembly. Despite the broad PKC isotype specificity of the inhibitors used, these data suggest that the two TJ proteins are differently regulated by PKC isotypes or subfamilies. As Gö 6983 uniquely affects aPKC (particularly PKCz) and we find that both PKCd and z relocate upon activator treatment to co-localise partially with the TJ proteins in isolated ICMs, we suggest that at least PKCd and z may play a central role in regulating TJ membrane assembly.

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Rat brain glial cells in primary culture and subculture contain the δ, ϵ and ζ subspecies of protein kinase C as well as the conventional subspecies

Cited by 31 publications

References 19 publications

Protein expression of the α, γ, δ and ε subspecies of protein kinase C changes as C6 glioma cells become contact inhibited and quiescent in the presence of serum

Protein expression of the α, γ, δ and ε subspecies of protein kinase C changes as C6 glioma cells become contact inhibited and quiescent in the presence of serum

Translocation of protein kinase C‐βII in astrocytes requires organized actin cytoskeleton and is not accompanied by synchronous RACK1 relocation

PKC signalling regulates tight junction membrane assembly in the pre-implantation mouse embryo

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