Etta Livneh

Isolation and characterization of PKC-L, a new member of the protein kinase C-related gene family specifically expressed in lung, skin, and heart.

We have isolated and characterized a new human cDNA, coding for a protein kinase, related to the protein kinase C (PKC) gene family. Although this protein kinase shares some homologous sequences and structural features with the four members of the PKC family initially isolated (alpha, beta I, beta II, and gamma), it shows more homology with the recently described PKC-related subfamily, encoded by the cDNAs delta, epsilon, and zeta. The transcript for this gene product, termed PKC-L, is most abundant in lung tissue, less expressed in heart and skin tissue, and exhibited very low expression in brain tissue. Thus, its tissue distribution is different from that described for other mammalian members of the PKC gene family, their expression being enriched in brain tissues. PKC-L is also expressed in several human cell lines, including the human epidermoid carcinoma line A431. The ability of phorbol esters to bind to and stimulate the kinase activity of PKC-L was revealed by introducing the cDNA into COS cells.

The Drosophila EGF receptor gene homolog: Conservation of both hormone binding and kinase domains

Chicken v-erB probe was used to isolate a unique clone of Drosophila melanogaster DNA. It maps by in situ hybridization to position 57F on chromosome 2. A complete nucleotide sequence of the coding region has been obtained. The putative Drosophila EGF receptor protein is similar in overall organization to the human homolog. It shows three distinct domains: an extracellular putative EGF binding domain, a hydrophobic transmembrane region, and a cytoplasmic kinase domain. The overall amino acid homology is 41% in the extracellular domain and 55% in the kinase domain. Two cysteine-rich regions, a hallmark of the human ligand-binding domain, have also been conserved. Fusion of the coding sequences of the kinase and extracellular domains generating the receptor gene must have occurred over 800 million years ago.

Mutations in the cytoplasmic domain of EGF receptor affect EGF binding and receptor internalization.

Binding of epidermal growth factor (EGF) to its receptor results in a cascade of events that culminate in cell division. The receptor is present on the cell surface in two forms of high and low affinity binding for EGF. EGF binding activates the receptors intracellular tyrosine kinase activity and subsequently causes the receptor to be rapidly internalized into the cell via clathrin‐coated pits. We have cloned the EGF receptor cDNA into a retroviral expression vector and made mutations in vitro to investigate the function of different receptor domains. Deletion of cytoplasmic sequences abolishes high but not low affinity sites as well as impairing the ability of the protein to internalize into cells. Thus, cytoplasmic sequences must be involved in the regulation of high affinity sites and are required for EGF‐induced receptor internalization. A four amino acid insertion mutation at residue 708 abolishes the protein‐tyrosine kinase activity of the immunoprecipitated receptor. However, this receptor mutant exhibits both the high and low affinity states, internalizes efficiently and is able to cause cells to undergo DNA synthesis in response to EGF. Another four amino acid insertion mutation (residue 888) abolishes protein‐tyrosine kinase activity, high affinity binding, internalization and mitogenic responsiveness. Finally, a chimaeric receptor composed of the extracellular EGF binding domain and the cytoplasmic v‐abl kinase region transforms Rat‐I cells. This chimaeric receptor possesses intrinsic protein tyrosine kinase activity which cannot be regulated by EGF. Moreover, EGF fails to induce the internalization of the chimaeric receptor.

The protein kinase C-related PKC-L(eta) gene product is localized in the cell nucleus.

The tumor promoters phorbol esters are thought to induce changes in cell growth and gene expression by direct activation of protein kinase C (PKC). However, the molecular mechanisms by which PKC molecules transduce signals into the cell nucleus are unknown. In this study, we provide evidence for a direct target for phorbol esters in the nucleus. We demonstrate that the new PKC-related family member, PKC-L, recently isolated by us, is expressed specifically in the cell nucleus. Localization of PKC-L in the cell nucleus is shown both by immunofluorescence staining and by subcellular fractionation experiments of several human cell lines, including the human epidermoid carcinoma line A431. Treatment of these cells by phorbol esters does not induce the down-regulation of PKC-L, in contrast to their effect on classical PKC family members. This is the only PKC isoenzyme described so far that resides permanently and specifically in the cell nucleus. PKC-L may function as an important link in tumor promoting, e.g., as a nuclear regulator of gene expression that changes the phosphorylation state of transcriptional components such as the AP-1 complex.

Release of a phorbol ester-induced mitogenic block by mutation at Thr-654 of the epidermal growth factor receptor.

The tumor promoter phorbol ester (TPA) modulates the binding affinity and the mitogenic capacity of the epidermal growth factor (EGF) receptor. Moreover, TPA-induced kinase C phosphorylation occurs mainly on Thr-654 of the EGF receptor, suggesting that the phosphorylation state of this residue regulates ligand-binding affinity and kinase activity of the EGF receptor. To examine the role of this residue, we prepared a Tyr-654 EGF receptor cDNA construct by in vitro site-directed mutagenesis. Like the wild-type receptor, the mutant receptor exhibited typical high- and low-affinity binding sites when expressed on the surface of NIH 3T3 cells. Moreover, TPA regulated the affinity of both wild-type and mutant receptors and stimulated receptor phosphorylation of serine and threonine residues other than Thr-654. The addition of TPA to NIH 3T3 cells expressing a wild-type human EGF receptor blocked the mitogenic capacity of EGF. However, this inhibition did not occur in cells expressing the Tyr-654 EGF receptor mutant. In the latter cells, EGF was able to stimulate DNA synthesis even in the presence of inhibitory concentrations of TPA. While phosphorylation of sites other than Thr-654 may regulate ligand-binding affinity, the phosphorylation of Thr-654 by kinase C appears to provide a negative control mechanism for EGF-induced mitogenesis in mouse NIH 3T3 fibroblasts.

An insertional mutant of epidermal growth factor receptor allows dissection of diverse receptor functions.

Cultured NIH‐3T3 cells devoid of endogenous EGF‐receptors were transfected with cDNA constructs encoding normal human EGF‐receptor and with a construct encoding an insertional mutant of the EGF‐receptor containing four additional amino acids in the kinase domain after residue 708. Unlike the wild‐type receptor expressed in these cells which exhibits EGF‐stimulatable protein tyrosine kinase activity, the mutant receptor lacks protein tyrosine kinase activity both in vitro and in vivo. Despite this deficiency the mutant receptor is properly processed, it binds EGF and it exhibits both high and low affinity binding sites. Moreover, it undergoes efficient EGF‐mediated endocytosis. However, EGF fails to stimulate DNA synthesis and is unable to stimulate the phosphorylation of S6 ribosomal protein in cells expressing this receptor mutant. Hence, it is proposed that the protein tyrosine kinase activity of EGF‐receptor is essential for the initiation of S6 phosphorylation and for DNA synthesis induced by EGF. However, EGF‐receptor processing, the expression of high and low affinity surface receptors and receptor internalization, require neither kinase activity nor receptor autophosphorylation. Interestingly, phorbol ester (TPA) fails to abolish the high affinity state and is also unable to stimulate the phosphorylation of this receptor mutant. This result is consistent with the notion that kinase‐C phosphorylation of EGF‐receptor is essential for the loss of high affinity EGF‐receptors caused by TPA.

Deletions in the regulatory or kinase domains of protein kinase C-α cause association with the cell nucleus☆

We have constructed expression plasmids carrying protein kinase C (PKC) cDNAs with deletions in the coding region. Two truncated molecules, consisting only of the kinase domain of PKC-alpha, were generated by removing parts of the cDNA coding for the regulatory region. Another mutant molecule was created by deleting 95 amino acids from the C-terminal part of the molecule. The full-length cDNA coding for PKC-alpha and its deletion constructs was expressed in COS cells. Using cell fractionation experiments and immunofluorescence staining, we demonstrate here that in contrast to the cytosolic localization of full-length PKC-alpha, the truncated forms, coding only for the kinase domain, were found exclusively in the cell nucleus. Further subfractionation of nuclei isolated from these transfected cells indicated partial association with the nuclear envelopes. Expression of the cDNA lacking the C-terminal part of the molecule in COS cells encoded a truncated molecule that was found both in the cytosol and in the nucleus. We also show that translocation of full-length PKC-alpha molecules to the cell nucleus occurred in response to phorbol ester treatment. Thus, it appears that accumulation of PKC-alpha in the nucleus results either by phorbol ester activation or by deletions of specific regions of the molecule. A molecular mechanism for the nuclear translocation of phorbol ester-activated PKC-alpha or its truncated molecules is suggested.

Regulation of Amyloid Precursor Protein Release By Protein Kinase C in Swiss 3T3 Fibroblastsa

Release of the amyloid precursor protein (APP) of Alzheimers disease from Swiss 3T3 fibroblasts was stimulated in a concentration‐dependent manner by phorbol 12‐myristate 13‐acetate. In fibroblasts overexpressing protein kinase Co (PKCα), the EC50 for this response was 7 nM, while in control cells the EC50 was 63 nM. The effect of PMA was inhibited by the PKC antagonist H‐7 in control cells, but not in cells that overexpressed PKCα. Basal release of APP was higher in cells that overexpressed PKCα, and was not affected by the phosphatase inhibitor okadaic acid, although this compound doubled APP release from control cells. The results suggest that PKCα regulates APP processing in mammalian cells. Alterations in the activity of PKC have been reported to occur in Alzheimers disease and might potentially contribute to abnormalities of APP metabolism characteristic of this disorder.

The expression of PDGF‐α but not PDGF‐β receptors is suppressed in Swiss/3T3 fibroblasts over‐expressing protein kinase C‐α

The generation and characterization of Swiss/3T3 cells which stably over‐express protein kinase C (PKC)‐α were previously described by us. In these cells over‐expression of PKC‐α reduced the expression of epidermal growth factor (EGF) receptor molecules [(1990) J. Biol. Chem. 265, 13290‐13296]. Here we show that the expression of PDGF‐α receptors, but not PDGF‐β receptors, was specifically decreased in these cells. Not only were the levels of PDGF‐α receptor mRNA transcript and protein significantly diminished in the PKC‐α over‐producing cells, but their ability to respond to short‐ and long‐term growth factor signals was appropriately compromised. This was reflected in a reduced tyrosine autophosphorylation signal in response to PDGF‐AA, as well as in decreased growth rates of PKC‐α over‐expressing cells when supplied with external PDGF‐AA. A similar decrease in PDGF‐α receptors was also demonstrated in parental Swiss/3T3 cells treated with phorbol esters. Our studies imply that PKC‐α is involved in a cellular mechanism suppressing the expression of PDGF‐α receptors in Swiss/3T3 cells. Hence, activation of PKC‐α or alterations in its cellular levels may affect, in turn, the expression of a specific set of cell surface receptors and their responses to external growth factors.

Specific photoalkylation of poly U and of poly A with 2-propanol and their messenger properties.

Ultraviolet light induced free radical alkylation of poly U or of poly A with 2‐propanol leads to the specific formation of 6‐(2‐hydroxy 2‐propyl) 5,6‐dihydro‐UMP or 8‐(2‐hydroxy 2‐propyl)‐AMP residues, respectively. The biochemical significance of the alterations in the modified polynucleotides was studied by testing their messenger activities in a cell free system of E. coli. Both photoalkylated poly U and poly A have reduced functional activity, with no change in functional specificity. This was demonstrated by inhibition of polymerization of phenylalanine and lysine directed by photoalkylated poly U and poly A, respectively, and by lack of misincorporation of other amino acids. The modification of poly U also caused the disappearance of misincorporation of serine that was found with the native polynucleotide.