Structural alteration in the MYB protooncogene and deletion within the gene encoding alpha-type protein kinase C in human melanoma cell lines.

Linnenbach, Alban J.; Huebner, Kay; Reddy, E. S. P.; Herlyn, Meenhard; Parmiter, Annette H.; Nowell̀, Peter C.; Koprowski, Hilary

doi:10.1073/pnas.85.1.74

Cited by 56 publications

(26 citation statements)

References 57 publications

(30 reference statements)

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“…Our results demonstrate that, in WM-1617 cells, PKCε is the much more prominent isoform than PKCα, and that PKCβ and PKCγ are essentially absent. This is consistent with the reported reduction or absence of these isoforms in different melanoma lines (Becker et al, 1990;Gilhooly et al, 2001;Linnenbach et al, 1988;Oka and Kikkawa, 2005;Oka et al, 1996). Based on our previous results (Mikule et al, 2003) and the data on PKC isoform abundance shown here, the effect of 12(S)-HETE is likely to be mediated by PKCε.…”

Section: Pkc Activation and Cell Detachmentsupporting

confidence: 93%

Dynamic adhesions and MARCKS in melanoma cells

Estrada-Bernal

Gatlin

Sunpaweravong

et al. 2009

Journal of Cell Science

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Cell motility necessitates the rapid formation and disassembly of cell adhesions. We have studied adhesions in a highly motile melanoma cell line using various biochemical approaches and microscopic techniques to image close adhesions. We report that WM-1617 melanoma cells contain at least two types of close adhesion: classic focal adhesions and more extensive, irregularly shaped adhesions that tend to occur along lamellipodial edges. In contrast to focal adhesions, these latter adhesions are highly dynamic and can be disassembled rapidly via protein kinase C (PKC) activation (e.g. by eicosanoid) and MARCKS phosphorylation. MARCKS overexpression, however, greatly increases the area of close adhesions and renders them largely refractory to PKC stimulation. This indicates that nonphosphorylated MARCKS is an adhesion stabilizer. Unlike focal adhesions, the dynamic adhesions contain α3 integrin and MARCKS, but they do not contain the focal adhesion marker vinculin. Overall, these results begin to define the molecular and functional properties of dynamic close adhesions involved in cell motility.

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Section: Pkc Activation and Cell Detachmentsupporting

confidence: 93%

Dynamic adhesions and MARCKS in melanoma cells

Estrada-Bernal

Gatlin

Sunpaweravong

et al. 2009

Journal of Cell Science

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“…Interestingly, alterated forms of PKC␣ have been observed in two cell lines. A smaller-than-expected PKC␣ was found in a small lung carcinoma cell line (57 kDa instead of 80 kDa), probably resulting from an aberrant posttranslational processing of the protein (18), and a tumor-specific deletion within the gene encoding PKC␣ was found in a primary melanoma cell line (23).…”

Section: Discussionmentioning

confidence: 99%

A Single Point Mutation in the V3 Region Affects Protein Kinase Cα Targeting and Accumulation at Cell-Cell Contacts

Vallentin

Joubert

2001

Molecular and Cellular Biology

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Given the importance of intercellular adhesion for many regulatory processes, we have investigated the control of protein kinase C␣ (PKC␣) targeting to the cell-cell contacts. We have previously shown that, upon treatment of the pituitary cell line GH3B6 with thyrotropin-releasing hormone (TRH) or phorbol 12-myristate 13-acetate (PMA), human PKC␣ (hPKC␣) is selectively targeted to the cell-cell contacts (42). Here we show that the D294G mutation of hPKC␣, previously identified in a subpopulation of human tumors, induces the loss of this selective targeting. The D294G mutant is instead targeted to the entire plasma membrane, including the cell-cell contacts, and the duration of the first rapid and transient translocation induced by TRH (42) is longer than that of the wild-type enzyme (93.3 versus 22.5 s), coinciding with the duration of the [Ca 2؉ ] i increase. We found that in the presence or absence of PMA, RACK1 is never localized at the cell-cell contacts nor was it coimmunoprecipitated with hPKC␣ wild type or the D294G mutant. In contrast, PMA treatment or long-term TRH stimulation resulted in the presence of F-actin and ␤-catenin at the cell-cell contacts and their exclusion from the rest of the plasma membrane. Upon disruption of the F-actin network with phalloidin or cytochalasin D, wild-type hPKC␣ translocates but did not accumulate at the plasma membrane and ␤-catenin did not accumulate at the cell-cell contacts. In contrast, the disruption of the F-actin network affected neither translocation nor accumulation of the D294G mutant. These results show that the presence of PKC␣ at the cell-cell contacts is a regulated process which depends upon the integrity of both PKC␣ and the actin microfilament network.Several years ago, we have shown that in a cell subpopulation of human pituitary and thyroid tumors, protein kinase C␣ (PKC␣) bore a point mutation at position 294, resulting in the substitution of an aspartic acid by a glycin (2, 31). The analysis of the biochemical properties of the D294G mutant and of the phenotype of embryonic fibroblasts stably transfected with it revealed a selective loss of recognition of substrates having characteristics of anchoring proteins (32) and a dramatic decrease in the dependence on serum growth factors for proliferation (3). In Rat6 fibroblasts stably transfected with human PKC␣ (hPKC␣) or its mutant and treated with phorbol 12-myristate 13-acetate (PMA) for 1 h, the D294G mutant localized in the lysosome compartment (unpublished data), whereas wild-type hPKC␣ (hPKC␣-wt) localized at the plasma membrane but not selectively at cell-cell contacts (3). Fibroblasts and epithelial cells are very different in many features. We therefore changed our model to the GH3B6 epithelial pituitary cell line. In this cell line, we found that PKC␣ is selectively targeted to the cell-cell contacts upon thyrotropinreleasing hormone (TRH) or PMA stimulation (42). To our knowledge, there is only one other study reporting on the presence of PKC␣ at the cell-cell contacts during spontaneous or...

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“…Several lines of evidence suggest that this might be the case. For example, a tumor-specific deletion in the PKCa gene was recently found in a malignant melanoma primary cell line (16), suggesting a possible link with tumorigenicity. Also, the c-raf gene, which is the cellular counterpart of the oncogene v-raf and which has been identified as an oncogene carrying serine and threonine kinase activity (18), was found to have a cysteinerich region in the N-terminal region.…”

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confidence: 99%

A protein kinase C cDNA without the regulatory domain is active after transfection in vivo in the absence of phorbol ester.

Muramatsu¹,

Kaibuchi²,

Arai³

1989

Mol. Cell. Biol.

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We constructed mutant protein kinase C (PKC) cDNAs which expressed PKC activity in vivo in the absence of phorbol ester activation. A hybrid PKC gene, PKAC, was constructed by substituting the coding region for the N-terminal 253 amino acids of PKCca with the N-terminal 17 amino acids of the cyclic AMP-dependent protein kinase catalytic subunit (PKA). A truncated PKC gene, APKCIB, lacking the coding region for amino acid positions 6 to 159 of PKCD was also constructed. These mutant kinase genes expressed under the control of the SRa promoter activated the c-fos gene enhancer in Jurkat cells and initiated maturation of Xenopus laevis oocytes. Phorbol ester binding activity was absent in both constructs but was preserved in another hybrid gene, PKCA, which was composed of the coding region for 1 to 253 amino acids of PKCa at the N-terminal side and the coding region for 18 to 350 amino acids of PKA at the C-terminal side. These results indicate that elimination of the regulatory domain of PKC produces constitutively active PKC that can bypass activation by the phorbol ester. APKCI3, in synergy with a calcium ionophore, was capable of activating the interleukin 2 promoter, indicating that cooperation of PKC-dependent and calcium-dependent pathways is necessary for activation of the interleukin 2 gene.Various hormones, growth factors, and neurotransmitters are known to elicit inositol phospholipid breakdown to produce diacylglycerol (DG) and inositol 1,4,5-trisphosphate in the target tissues (1, 19). DG and inositol 1,4,5-trisphosphate serve as second messengers for activation of protein kinase C (PKC) and calcium mobilization, respectively (1, 19). Phorbol ester and membrane-permeable DG have been used to study the function of PKC because they selectively activate PKC without inducing calcium mobilization in intact cells (25). By use of these agents, it has been shown that PKC activation and calcium mobilization are essential elements of cellular responses such as release reaction (12, 15), cell proliferation (11,30), and gene expression (3, 13). Since the phorbol ester induces the down regulation of PKC (24) and membrane-permeable DG is easily converted to the corresponding phosphatidic acid by the action of DG kinase (25), the activation of PKC by these agents appears to be transient in most cells, and therefore, it is inconvenient to use it in certain studies.In early studies of PKC, it was shown that partial proteolysis of this enzyme by a calcium-dependent protease generated a 51-kilodalton component with catalytic activity but without the requirement for activators such as phospholipid, phorbol ester, and calcium ion (25). The other product of proteolysis has been shown to bind to a complex of phospholipid, phorbol ester, and calcium ion (7). These results suggest that PKC might be composed of separate regulatory and catalytic domains.Recently, based on amino acid sequences of PKC deduced from cDNAs cloned from various species (4,9,14,(20)(21)(22), several domains of the PKC molecule have been delineated. The...

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Structural alteration in the MYB protooncogene and deletion within the gene encoding alpha-type protein kinase C in human melanoma cell lines.

Abstract: A correlative study was done to determine possible relationships between nonrandom aberrations in chromosomes 1, 6, and 7 occurring in human cutaneous malignant melanoma and the structure of oncogenes as well as specific genes encoding growth factors and growth factor receptors.

Cited by 56 publications

References 57 publications

Dynamic adhesions and MARCKS in melanoma cells

Dynamic adhesions and MARCKS in melanoma cells

A Single Point Mutation in the V3 Region Affects Protein Kinase Cα Targeting and Accumulation at Cell-Cell Contacts

A protein kinase C cDNA without the regulatory domain is active after transfection in vivo in the absence of phorbol ester.

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