Potentials of PKC in cancer development and therapeutic outcomes
H) Epigenetic agents: These drugs are designed to effect chromatin modification of tumor suppressor genes or oncogenes. Vorinostat is known to inhibit the enzymatic activity of histone deacetylases
I) Immunomodulatory agents: These agents boost the ability of the immune system to fight cancer
1.9 Structural and Biochemical Details of PKC
1.9.1 Structure of Protein kinase C: The PKC family consists of several isozymes which share a common 3-Dimensional fold: a kinase domain present on the C-terminal and a flexible hinge segment
extracellular matrix with its numerous components pose a big hurdle for cancer cell movement.
Degradation of the basement membrane is necessary for primary tumors to invade and gain access to the circulatory system. Invading cancer cells accomplish this by the secretion of metallo-protease enzymes (MMPs) which serve to degrade the basement membrane (BM) and extracellular matrix (ECM) proteins. These cleavage process serve to liberate and mobilize various growth factors that remain tethered in inactive form in the ECM. Invasive carcinomas recruit a variety of stromal cells that also secrete different MMPs (Vong and Kalluri, 2011). The recruited stromal cells consists of macrophages, fibroblasts and mast cells. An important metallo-protease, MTI-MMP is directly tethered to the plasma membrane of cancer cells (Yao et al., 2006). MTI-MMP is extensively used by invading cancer cells like a scissor to cleave critical ECM components as they slowly move through the ECM. Again, PKC has been found to stimulate MMPs through EGFR signalling in breast cancer cells (Lin et al., 2008; Soto-Guzman et al., 2010). After, gaining access to the lumen of blood vessels, invading cancer cells migrate for newer locations to establish colonies in a chemotaxis driven process.
During metastasis, stimuli such as growth factors, cytokines, etc. play a vital role in the induction of chemotaxis of malignant tumor cells. Activation of PKC has been found to activate components of the chemotactic pathways in MDAMB-231, T47-D, and MCF-7 breast cancer cells. (Sun et al., 2005;
Wang et al., 2008). Thus, PKC plays important roles in the invasion and metastasis of cancer cells.
Carcinogens catechol and hydroquinone promotes tumor metastasis via activation of PKC in lung carcinoma cells (Gopalakrishna et al., 1994). PKC is found to affect at the level of chromatin modulation to activate metastasis in cancer cells (Lin et al., 2012). PKC can also serve as a therapeutic target to halt metastasis as suppression of PKC-α halts metastasis in ovarian cancer cells (Jiang et al., 2005). Hence, understanding the crucial role of PKC in cancer induction and metastasis opens up avenues for drug development.
Chapter 1|27 phosphorylation of another motif in the kinase domain, the activation loop (shown in purple in Figure 1.14-D) by the PDK-1 kinase. The V5 region present at the carboxy-terminal region is the site for important phosphorylations by the mTORC2 complex. These phosphorylation events are crucial for the development of the final mature & functionally active form of the PKC.
The pseudo-substrate region forms the extreme end of the N-terminal region. It occupies the substrate binding site of the Kinase domain in the absence of any agonist/ligand of the C1 domains.
Presence of agonists of the C1 domains direct the C1 regions away from the kinase domain towards the cellular membranes. As a result, the substrate binding site of the kinase domain is relieved of the pseudo-substrate region. Perhaps, the early region (residues 619-633 in PKC-βII) of carboxy tail known as the NFD-helix region (shown in red in Figure 1.13) is a very interesting regulatory element of PKC activation. The NFD-helix has an important amino acid residue, Phe 629 (in PKC-βII) that is important to provide stability to ATP at the ATP binding site. In the absence of any agonist, the NFD- helix remains bound to the C1 domain in the agonist binding site, and the Phe 629 is prevented from contacting the ATP binding site. However, agonist binding to C1 domain releases the NFD-helix which is now in an elongated state. In this state, the Phe 629 residue is able to contact the ATP binding
Figure 1.13: Structure of PKC-βII (PDB ID: 3PFQ). Kinase domain is shown in cyan along with C1b domain (green) and C2 domain (blue).
site and promote ATP binding. Although, this model is described for PKC-βII, this is similar for other PKC isozymes as well.
1.9.2 Protein Kinase C and its different isoforms: Protein Kinase C (PKC) are serine-threonine kinases that exists as a family of isozymes consisting of 10 members discovered till date in mammalian cells and belong to the family of AGC kinases (Pearce et al., 2010). The 10 members are grouped into 3 classes based on their domain composition and order of individual domains (Figure 1.15). The first class are the conventional PKCs (cPKCs) comprising of PKC-α, PKC-βI, PKC-βII and PKC- γ.(Nishizuka, 1995). Amongst the cPKCs, PKC-βI and PKC-βII are the alternatively spliced versions of the same gene. The second class are the novel PKCs (nPKCs) which comprise of PKC-δ, PKC-ε, PKC-η and PKC-θ. The final classes are called as atypical PKCs (aPKCs) and they have two family members PKC-ζ and PKC-ι/λ. The C1 domain is an important constituent of protein structure for both cPKCs and nPKCs as it is an important regulatory domain. Likewise, the C2 domain is also a regulatory domain but is functionally active only for cPKCs. A functionally redundant form of C2 domain is present in nPKCs which is altogether absent in the atypical isozymes. Instead, aPKCs
Figure 1.14: General Structure of PKC (A) Illustration of full structure of PKCα (B) C1 domain of PKC- α (C) C2 domain of PKC-α (D) Kinase domain of PKC-βII (PDB ID: 3PFQ).
Chapter 1|29 contain a unique domain, the PB1 domain which serves to provide regulatory function. All PKC isozymes have a central hinge region (Figure 1.15) which works as a connector to the regulatory subunits towards the N- terminal side and the kinase subunit towards the C-terminal side.
PKC isozymes activation is regulated by various ligand-protein and protein-protein interactions. Early studies indicated that after activation, much of the active enzyme was present in the particulate fraction rather than the cell soluble fraction (Takai et al., 1979). The C1 domain of cPKCs and nPKCs were later found to bind to lipid messengers which speculated their translocation primarily to plasma membranes. Later, it was revealed that many of these isozymes translocate to many different sub-cellular organelle membranes such as the mitochondrial membrane (Churchill et al., 2005), the Golgi apparatus (Lehel et al., 1995), the endoplasmic reticulum (Qi and Mochly-Rosen, 2008), on cell- cell contacts (Vallentin et al., 2001), contractile elements and even inside the nucleus (Disatnik et al., 1994). Receptor for active C Kinases (RACKs) are a class of scaffold proteins that are known to interact actively to different PKC isozymes (Mochly-Rosen et al., 1991).
Thus, different PKC isozymes are localized to a subset of substrates in different cell-types and under different physiological conditions which results in differential isozyme-selective functions. A detailed review of the individual domains will further clarify the functional attributes of each class of PKC isozymes in the cellular microenvironment.
1.9.3 C1 domain: The C1 domain is the principal regulatory domain of cPKCs and nPKCs as it binds