Protein Kinase C
Protein Kinase C (PKC) serves an important role in a cell's response to external stimuli. It's crucial in activities such as cell differentiation, proliferation and geometry. PKC is not a single enzyme but a family of serine/threonine kinases with as much as 10 isoforms, each with its own activation needs. Activation of PKC occurs when diacylglycerols are produced as a result of activation of plasma membrance receptors coupled to phospholipase C.
The structure of PKC is in the form of a single polypeptide with a regulatory N-terminal domain and a catalytic C-terminal domain. Within each isosomes of PKC there exists 4 regions named C1-C4. Each region has a specialized binding activity designated. C1 contains a cysteine rich motif which allows binding with diacylglycerol. Within the C2 region is a recognition site for acidic lipids and in some isosomes a site for calcium. Respectively C3 and C4 allows ATP and substrates to bind. PKC exists in the inactivated form, but the inactivated form is not distributed freely in the cytosol but are localized to specific sites in the cell. In it's inactive state PKC has the pseudosubstrate domain attached to the catalytic domain. Once stimulated PKC moves towards the plasma membrane where C1 binds to DAG and C2 interacts with phosphatidylserine. After this happens it causes the dislocation of the pseudosubstrate domain from the catalytic site and hence PKC becomes activated.
Research in Shape Alteration
PKC activators such as DAG and phorbol esters are known to stimulate several functions in neutrophils including shape changes. An early study by Keller et al, 1990, showed that inhibition of PKC's activity affected shapes of neutrophils. Locomotion and actin plymerization are however, suppressed with PKC activators. This study emphasizes the involvement of PKC in shape alterations in cells by demonstrating H-7 (a PKC inhibitor) caused neutrophils to go through shape alterations. Cells treated with H7 remained round instead of having polarity seen in neutrophils responding to chemotactic signals.
In a later study, Belusa at al, 1997, showed PKC's involvement in cell structure alteration by mutating and enzyme which PKC activates. The enzyme Na+, K+ -ATPase creates a Na+ gradient within the transmembrane that is important for the normal function of the ekaryote cells. The enzyme can either be phosphorylated by PKA or PKC on particular sites within the enzyme structure. PKC phosphorylation site SER23 has been identified as the major site for this process. In this particular study by Belusa et al 1997, SER23 was mutated by being replaced with alanine and the results where observed. A few factors were observed in particular, 86Rb+ uptake, intracellular Na+ and pH. Results indicated that when cells were saturated with Na+ concentrations of 10mM and 70mM, the PKC activator, PDBu inhibited the enzyme's activity by roughly 24±7% at 70mM and 27±5% at 10mM in wild type rats. In contrast, rats with the mutated SER23 site showed no effect by PDBu at both concentration of Na+ saturation. Rb uptake was affected by PDBu in mutatated SER23 enzymes where in wild type, there were reduction in it's uptake. This particular experiment demonstrated that cells transfected with a mutated SER23 enzyme were no longer able to respond to PKC, thus the reduction in RB uptake. Furthermore, the mutated cells were no longer able to grow elongated extensions and remained round in shape while the wild types grew elongated extensions. With outcomes demonstrated in this study, one could see that the morphological change is attributed to by the inhibition of PKC activities and because PKC exists in isoforms, further studies is required in tracing which particular pathway and which isoform is involved in it's effect with cell structure.
A more recent study involving fibrosarcoma cells, by Keller et al, 2000, revealed once again PKC's involvement in a cell structure alteration. This was done by using Phorbol myristate acetate (PMA), a PKC activator. Upon stimulation by PMA, fibrosarcoma cells HT1080, showed a more crescent like shape as oppose to cells having elongation and irregularity. This change in cell structure is associated with the reorganization of the actin cytoskeleton. When stimulated by PMA, cytosol isoforms of PKC shifts towards the membrane fraction. The study also showed that upon activation of PKC and shape changes, the cancerous cells reduced in it's invasiveness. PKC could potentially have a therapeutical use in cancerous cells since it is also seen in effects with many cell lines.
- Belusa, R., Wang, Z., Matsubara, T., Sahlgren, B., Dulubova, B., Nairn, A., et al. (1997). Mutation of the Protein Kinase C Phosphorylation Site on Rat a1 Na1,K1-ATPase Alters Regulation of Intracellular Na1 and pH and Influences Cell Shape and Adhesiveness. The Journal of Biological Chemistry, 272(32), 20179-20184.
- Keller, H. U., Niggli, V., & Zimmermann, A. (1990). The protein kinase C inhibitor H-7 activates human neutrophils: effect on shape, actin polymerization, fluid pinocytosis and locomotion. Journal of Cell Sciences, 96(1), 99-106.
- Keller, H. U., Hunziker, I. P., Sordat, B., Niggli, V., & Sroka, J. (2000). Protein kinase C isoforms involved in regulation of cell shape and locomotion of human fibrosarcoma HT1080 cells. International journal of cancer , 88(2), 195-203.
- Steinberg, S. F. (2008). [Review of the book Structural Basis of Protein Kinase C Isoform Function]. Physiology Reviews, 88, 1341-1378.
- Chen, S.J. et al. (1993) Studies with synthetic peptide substrates derived from the neuronal protein neurogranin reveal structural determinants of potency and selectivity for protein kinase C. Biochemistry 32, 1032.
- It has a double membrane.
- The shape of the nucleus is defined by the cytoskeleton.
- Structures within the nucleus are: Cajal bodies, chromatin.
- mRNA is synthesized within the nucleus.
The difference between SER and RER is confusing. What is the function of the ribosomes attached to the RER? So what structure within the cytosol actually packages the proteins into vesicles ready to be exocytosed?
One process which might require a lot of energy would be cell division. Another would be a sperm requiring energy to allow mobility of the flagella.
L-cam is a liver adhesion molecule, which utilizes calcium to allow cell to cell adhesion. I-cam is an intercellular adhesion molecule and is expressed intramembranously in leukocytes and endothelial cells at low concentration. However when cytokine signals are detected, the expression of these adhesion molecules will increase dramatically. N-cam molecules are neural cell adhesion molecule. Although the name suggests their existence on neural cells only, they are in fact found in skeletal muscle cells and NK cells. They bind homophillically and have the ability to memorize and also allows neural outgrowth.
Two methods of confocal microscope
Laser and Spinning disc
The s phase stands for the synthesis phase of the cell cycle. It is during this phase that DNA synthesis occurs.