PKC epsilon-inducible mRNA

Protein kinase C (PKC) is a phospholipid-dependent serinehreonine kinase family consisting of at least 11 related isozymes. These PKC isozymes play important roles in signal transduction pathways. The exact function of each PKC isozyme is not known at present. PKC epsilon seems to have the function of an oncogene, although the exact role of this isozyme is also not known at present. In order to elucidate the role of PKC epsilon we investigated whether this isozyme leads to alterations in the expression of other genes. We cloned a constitutively active PKC epsilon (alanine in position 129 is replaced by glutamic acid) in a tetracycline-inducible vector and stably transfected HeLa cells with this construct. The cells were screened and a clone which expresses constitutively active PKC epsilon in presence of doxycycline, but not in its absence, was isolated. Gene filter microarrays were hybridized with 33P-labelled cDNA derived from total RNA of uninduced HeLa control cells and of HeLa cells in which the expression of constitutively active PKC epsilon was induced with doxycycline. We found that expression of constitutively active PKC epsilon among other alterations induces the expression of an EST (PKC epsilon-inducible EST mRNA). The upregulated sequence is an EST from a mRNA from which a high quality sequence of 368 bases from the 3end is known. This sequence is not related to any known gene. It is intended to perform investigations into this mRNA and the encoded protein. The aim of the proposed project is (1) to control whether overexpression of constitutively active PKC delta, eta and theta also leads to to overexpression of the PKC epsilon-inducible mRNA (PKC alpha and zeta do not), (2) to screen different human tissues for the expression of the PKC epsilon-inducible EST mRNA, (3) to obtain a full length clone of the PKC epsilon-inducible EST mRNA and (i) sequence it and (ii) to transfect and overexpress full length PKC epsilon-inducible EST mRNA, (4) to generate an antibody directed against the PKC epsilon inducible EST-encoded protein, to investigate where the protein is located in the cell and whether it colocalizes with PKC epsilon, (5) to find proteins interacting with the PKCeEST-encoded protein by coimmunoprecipitation and by the Yeast Two-Hybrid system.

Protein kinase C is a family of 10 related proteins (denominated alpha, beta I, beta II, gamma, delta, eta, epsilon, theta, iota und zeta). These proteins seem to play a role in different diseases such as allergy, asthma, rheumatoid arthritis, AIDS, Alzheimers disease, multiple sclerosis, hypertension, cardiac hypertrophy, atherosclerosis, diabetes and cancer. Ruboxistaurin is an inhibitor of protein kinase C beta I and II. In clinical tests the compound showed, that it can prevent diabetic complications (diabetic foot is a combination of blood vessel and neurologic damage, diabetic polyneuropathy is a damage of neurons, diabetic retinopathy is a disease of small blood vessels in the eye, diabetic nephropathy is damage of the kidneys). The exact function of the 10 protein kinase C proteins is not known at present. We want to elucidate the function of protein kinase C epsilon. It might be involved in carcinogenesis, cardiac failure, alcohol consumption and pain sensitivity. Knowledge of the exact function of protein kinase C epsilon would be important for the development of potential new drugs for the treatment of the diseases mentioned. In order to explain the role of protein kinase C epsilon in the cell, we previously investigated which genes it can activate or down-modulate. We found that protein kinase C epsilon increases the expression of one gene and down-modulates 30 others. Only parts of these genes were so far. In this project we have investigated one protein kinase C-upregulated (AA100674) and two down-modulated, unknown genes (C6orf69 and RHOBTB3). We could not establish the full length of the upregulated gene. In the meantime two files were found in the databases which indicate that it is involved in the opening of DNA or in the folding of proteins. From the two down-modulated sequences we obtained full length genes and the composition of the proteins. Both contain a domain (BTB/POZ domain) that seems to be involved in the regulation of genes or in the interaction with other proteins. This is an indication that these proteins are involved in regulation mechanisms in cells. Elucidation of their function could yield knowledge of the function of protein kinase C epsilon. On the long run the aim is to find out whether these proteins are involved in signal transduction within cells and whether inhibitors can be employed for therapies of above mentioned diseases.