Protein Kinase C

Protein kinase C is a family of serine threonine kinases which are believed to play important roles in the regulation of mammalian growth, differentiation and apoptosis. During activation PKC isoforms generally translocate from the cytosol to the membrane or other cellular compartments which provides a potential measure of the activation state of the isoform (Figure 1). Individual isoforms are targeted to specific intracellular sites by receptors for activated PKC (Known as RACK = "receptor for activated C kinase") for the activated form of the isoform  which is probably necessary for appropriate and specific signaling.

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Figure 1: Protein kinase C signaling

 

PKC is activated by the fatty acid derivative diacylglycerol, with Ca2+ acting as a cofactor for some isoforms. Activated PKC is targeted to different subcellular compartments by binding to receptors (RACK's) which are specific to each isoform.

Dr Hatton, working in the research group of Dr Didier Schmitt in Strasbourg, France, investigated the effect of microgravity on protein kinase C isoform signaling in monocytes and T-cells during the course of several Space Shuttle experiments from 1994 to 1997. These experiments were performed in the European Space Agency Biorack facility, which provided a 37˚C incubator, an on-board 1.g centrifuge for control experiments and glovebox for manipulating the experiment. The experiments were performed in a special cell culture unit, which was developed by the French space agency (CNES).

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(b) GCAK-1 cassette system showing multi-injector unit

(a) GCAK-2 cassette system

 

 

Figure 2: Cell culture system used for spaceflight experiment

 

The first experiment performed on STS-65 (IML-2) in 1994 showed that the intracellular distribution of total PKC varied with g-level in both the U937 human monocytic cell line and Jurkat human T-cell line. In microgravity the amount of PKC in the cytosol of the cell was lowest in microgravity, higher in the 1.g ground and highest on the ground 1.4g centrifuge. The in-flight 1.g centrifuge PKC cytosolic PKC levels were intermediate between microgravity and 1.g ground probably due to the exposure of these samples to microgravity prior to activation of the in-flight centrifuge.

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       (A) Jurkat cells                                                                       (B) U937 cells                     

Figure 3: The intracellular distribution of total PKC in Jurkat T-cell and U937 monocyte cell lines

 

The effect of microgravity on PKC mediated signaling in monocytes and T-cells was further explored in subsequent experiments in Biorack on STS-76, STS-81 and STS-84 (all missions which were part of the Shuttle-Mir program). In the STS-76 experiment <Link to Hatton et al 1999.pdf> total PKC per cell was elevated in U937 cells exposed to microgravity conditions for 3 days compared to 1.g ground conditions, while total PKC in 1.4g cultured cells was significantly lower than in any of the other g-levels.

In the STS-81 mission <Link to Hatton et al 2002.pdf> we investigated the effect of microgravity on the activation of selected PKC isoforms. U937 cells were stimulated with a phorbol ester which is a synthetic analogue of the endogenous PKC activator, diacylglycerol. Phorbol esters induce the translocation of PKC isoforms to the membrane and other cellular compartments. We observed that the translocation of PKC bII, d and e was inhibited in microgravity compared to 1.g ground conditions, while translocation of each isoform was enhanced in 1.4g hypergravity.

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Figure 4: PKC isoform quantity in the particulate fraction of (a) unstimulated U937 cells and (b) U937 cells stimulated for 10 minutes with PDBu, a phorbol ester. (c) The relative change in particulate PKC isoforms between unstimulated and stimulated U937 cells. Note the difference in the pattern of PKC redistribution in microgravity compared to 1.g, in particular the distribution of PKC delta.

 

In addition when we compared the relative change in PKC isoforms associated with the particulate fraction of the cell (membrane, insoluble cytoskeleton, nucleus) between unstimulated and phorbol ester stimulated U937 cells were observed that the pattern of redistribution of PKC isoforms in microgravity was markedly different to the other g-levels.

These experiments showed that PKC isoform activation is generally inhibited in microgravity, with individual isoforms showing different levels of sensitivity. Since individual PKC isoforms are believed to play distinct roles in the regulation of monocyte differentiation, alterations in the pattern of activation of each isoform could have important consequences for monocyte function under microgravity conditions.

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Figure 5: PKC isoforms fulfil distinct roles in the regulation of monocyte differentiation

 

Individual PKC isoforms appear fulfill distinct roles in monocytes. PKC b-II appears to be essential for differentiation, PKC d is implicated in programmed cell death (apoptosis), while PKC e may have an anti-apoptotic role.

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