Ectopic expression of a mutant form of PKCα originally found in human tumors: aberrant subcellular translocation and effects on growth control

Abstract

A point mutation in PKCa was originally discovered in a subpopulation of human pituitary tumors characterized by their invasive phenotype, and the same mutation was also seen in some thyroid neoplasms. To investigate the role of this mutation in tumorigenesis, normal and mutant human PKCa cDNAs were overexpressed in Rat6 embryo ®broblasts (R6). When extracts of R6 cells that expressed either the normal or mutant PKCa were assayed in the presence of calcium, phosphatidylserine and the phorbol ester TPA, for phosphorylation of either histone IIIS or the EGF-receptor peptide, both extracts gave similar results. However, the subcellular localization of the two proteins diered. Immunohistochemistry studies indicated that after treatment with TPA normal PKCa mainly translocated to the plasma membrane, but mutant PKCa translocated mainly to the perinuclear region and slightly to the nucleus. Furthermore, the cells that expressed the mutant PKCa displayed a decreased requirement for serum when compared to the cells expressing the normal human PKCa, and they formed small colonies in soft agar. By contrast, the cells expressing the normal human PKCa failed to form colonies in soft-agar. Thus, ectopic expression in rat ®broblasts of this mutant human PKCa sequence alters the growth properties of these cells and, when activated, the mutant PKCa displays aberrant intracellular translocation. Therefore, this mutation in PKCa could contribute to the process of tumor progression in certain human tumors.

The α isoform of protein kinase C mediates phorbol ester-induced growth inhibition and p21cip1 induction in HC11 mammary epithelial cells

“…Protein lysates were prepared from exponentially growing HC11 cells that overexpress PKCa (pBabePKCa), PKCb 1 (pBabe-PKCb 1 ) or vector control (pBabe-). The cells were either untreated (7) or treated with TPA for 6 h. Western blot analysis was then performed using an antibody speci®c for p21 cip1 translocated to the perinuclear region and the nucleus when the cells were treated with TPA, in contrast to the wild-type PKCa which did not translocate to the nucleus in this cell line (Alvaro, 1993;Alvaro et al, 1997). These ®ndings suggest that the residues that determine nuclear localization reside in the less conserved (variable) regions of PKCa.…”

“…To create the pUHD-PKCa plasmid pBabe-PKCa (Alvaro et al, 1997) was cut with EcoRI, and the human cDNA encoding PKCa was gel-puri®ed using the Qiaex kit (Qiagen, Chatsworth, CA, USA) and ligated into the EcoRI site of the pUHD 10-3 vector (Gossen and Bujard, 1992). To create the pUHD-PKCb 1 plasmid the RP58 plasmid (Housey et al, 1988) was digested with HindIII and NotI, and the rat cDNA encoding PKCb 1 was gel-puri®ed and then ligated into the pMT vector digested with HindIII and NotI, creating pMTPKCb 1 .…”

“…pMT-PKCb 1 was then digested with BamHI, and the fragment containing the PKCb 1 cDNA gel-puri®ed and ligated into the BamHI site of pUHD 10-3. The pBabe vector and pBabe-PKCa plasmids were previously described (Alvaro et al, 1997). To create pBabe-PKCb 1 the RP58 plasmid was digested with BamHI, and the rat cDNA encoding PKCb 1 was gel-puri®ed and ligated into the pBabe vector digested with BamHI, creating pBabe-PKCb 1 .…”

“…After treatment with TPA (100 ng/ml) for various times the slides were washed three times in PBS, ®xed in formaldehyde and prepared for immunohistochemistry as described elsewhere (Alvaro et al, 1997). The antibodies used were 5 mg/ml anti-PKC a (a nity puri®ed preparation from Gibco ± BRL) and a 1 : 1000 dilution of FITC-a-rabbit IgG (BioSource).…”

Defective IgG2a/2b Class Switching in PKCα−/− Mice

“…Overexpression of rPKC␣ in the epidermis of transgenic mice resulted in striking alterations of PMA-induced edema, infiltration of neutrophils, and expression of genes implicated in inflammation such as cyclooxygenase 2 and TNF-␣ (10). Conversely, ectopic recombinant expression of mutant PKC␣ inhibited bacterial LPS-induced cytokine production in macrophages (11). Along this line, and shown by overexpression or inhibition of PKC␣ in cell lines, PKC␣ regulates I ␤ kinase and NF-B also in T cells (12,13).…”