Identification of an Amino Acid Residue in the Protein Kinase C C1b Domain Crucial for Its Localization to the Golgi Network

Schultz, Anna Reister; Ling, Mia; Larsson, Christer

doi:10.1074/jbc.m313017200

Cited by 43 publications

(51 citation statements)

References 44 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…7D). Consistent with previous reports (Kajimoto et al, 2004;Schultz et al, 2004), the PKCδ ΔC1B mutant was defective in localizing at the Golgi complex (Fig. 7D).…”

Section: Pkcδ Targets Cell-cell Junctions and Cellular Membranes Throsupporting

confidence: 81%

Functional suppression of E-cadherin by protein kinase Cδ

Chen

2009

Journal of Cell Science

View full text Add to dashboard Cite

Journal of Cell Science 514 cell functions, including cell proliferation (Ashton et al., 1999;Kitamura et al., 2003), differentiation (Corbit et al., 1999;Pessino et al., 1995), apoptosis (Brodie and Blumberg, 2003;Kajimoto et al., 2004;Zhong et al., 2002) and tumor suppression (Lu et al., 1997;Reddig et al., 1999). Increasing evidence also indicates that PKCδ has a positive role in cell motility (Chen et al., 2007;Gliki et al., 2002;Iwabu et al., 2004;Li et al., 2003) and the metastatic potential of tumor cells (Kiley et al., 1999;Kruger and Reddy, 2003; Alonso-Escolano et al., 2006;Kharait et al., 2006;Villar et al., 2007). However, the role of PKCδ in intercellular junctions remains obscure. In this study, we aim to explore the role of PKCδ in cellcell junctions using Madin-Darby canine kidney (MDCK) cells as a model. Results Expression of GFP-PKCδ, but not GFP-PKCα, suppresses the homophilic interactions between the ectodomains of E-cadherins in MDCK cellsTo explore the role of PKC in cell-cell junctions, GFP-PKCδ and GFP-PKCα were stably expressed in MDCK cells (Fig. 1A). Expression of either construct in those cells did not alter their growth (data not shown) or their ability to form cell colonies within which the cells are in contact with each other (Fig. 1B). To examine their effect on adherens junctions and tight junctions, cells stably expressing GFP-PKCδ or GFP-PKCα were co-cultured with parental control MDCK cells and then grown to confluence. The cells were fixed and stained for adherens junctions and tight junctions with anti-E-cadherin and anti-ZO-1, respectively. Neither GFP-PKCδ nor GFP-PKCα affected tight junctions of MDCK cells (Fig. 1C,D). However, the fluorescence intensity of E-cadherin at cell-cell contacts in the cells expressing GFP-PKCδ, but not GFP-PKCα, was much weaker than that in adjacent parental MDCK cells (Fig. 1C,D).As the total and cell surface levels of E-cadherins were not altered by GFP-PKCδ (supplementary material Fig. S1), it is unlikely that the decreased fluorescence intensity of E-cadherin was due to suppression of E-cadherin expression. The rat monoclonal anti-Ecadherin (clone ECCD-2) used in Fig. 1C is known to recognize the extracellular domain of E-cadherin, rendering it possible that GFP-PKCδ causes a conformational change on the ectodomain of E-cadherin, which then prevents E-cadherins from detection by the ECCD-2 antibody. To clarify this, a mouse monoclonal antibody (clone 36) that recognizes the cytoplasmic portion of E-cadherins was used. The fluorescence intensity of E-cadherin with the clone 36 antibody did not show differences between control cells and PKCδ-overexpressed cells ( Fig. 2A), supporting the idea that the conformation of E-cadherins rather than their distribution or expression is affected by PKCδ. In addition, we found that the ability of the ECCD-2 antibody, but not the clone 36 antibody, to detect E-cadherin at cell-cell junctions relied on the presence of Ca 2+ in the culture medium, as demonstrated by Ca 2+ -switch assay (Fig. 2B), indicat...

show abstract

“…7D). Consistent with previous reports (Kajimoto et al, 2004;Schultz et al, 2004), the PKCδ ΔC1B mutant was defective in localizing at the Golgi complex (Fig. 7D).…”

Section: Pkcδ Targets Cell-cell Junctions and Cellular Membranes Throsupporting

confidence: 81%

Functional suppression of E-cadherin by protein kinase Cδ

Chen

2009

Journal of Cell Science

View full text Add to dashboard Cite

show abstract

“…7C,D). The C1B domain of PKCd has been shown to be crucial for its localization at the Golgi complex (Kajimoto et al, 2004;Schultz et al, 2004). As expected, the DC1B mutant did not localize at the Golgi (supplementary material Fig.…”

mentioning

confidence: 57%

Elevated expression of protein kinase Cδ induces cell scattering upon serum deprivation

Chen

Chan

Wang

et al. 2010

Journal of Cell Science

View full text Add to dashboard Cite

SummaryTumor metastasis might be evoked in response to microenvironmental stress, such as a shortage of oxygen. Although the cellular response to hypoxia has been well established, we know little about how tumors adapt themselves to deprivation of growth factor. Protein kinase Cd (PKCd), a stress-sensitive protein kinase, has been implicated in tumor progression. In this study, we demonstrate that elevated expression of PKCd in Madin-Darby canine kidney cells induces a scatter response upon serum starvation, a condition that mimics growth-factor deprivation. Serum starvation stimulates the catalytic activity and Y311 phosphorylation of PKCd through reactive oxygen species (ROS) and the Src family kinases. Mutation of PKCd at Y311 and Y322, both of which are phosphorylation sites for Src, impairs its activation and ability to promote cell scattering upon serum deprivation. Once activated by ROS, PKCd itself activates ROS production at least partially through NADPH oxidase. In addition, the c-Jun N-terminal kinase is identified as a crucial downstream mediator of ROS and PKCd for induction of cell scattering upon serum deprivation. We demonstrate that the C1B domain of PKCd is essential not only for its localization at the Golgi complex, but also for its activation and ability to induce cell scattering upon serum deprivation. Finally, depletion of PKCd in human bladder carcinoma T24 cells restores their cell-cell contacts, which thereby reverses a scattered growth pattern to an epithelial-like growth pattern. Collectively, our results suggest that elevated expression of PKCd might facilitate the scattering of cells in order to escape stress induced by growth-factor deprivation. (B)An equal amount of whole cell lysate from neomycin-resistant control MDCK cells (neo) and those stably expressing HA-PKCd or HA-FAK was analyzed by immunoblotting with anti-HA. (C)MDCK cells as described for A were allowed to grow as cell colonies in medium supplemented with 10% serum. Subsequently, they were maintained in medium with (+) or without (-) 10% serum and monitored by time-lapse video-microscopy. Representative micrographs at 0, 24, 30 and 36 hours are shown. Scale bars: 150mm. (D)Cell colonies formed by MDCK cells stably expressing HA-PKCd or HA-FAK were maintained in medium with (+) or without (-) 10% serum and monitored by time-lapse video-microscopy. Representative micrographs at 0, 24, 30 and 36 hours are shown. Scale bars: 150mm. (E)MDCK cells stably expressing GFP-PKCd were allowed to grow as colonies and then maintained in medium containing different amounts of serum. A cell colony was judged as a 'scattered' one when one third of the cells in it had lost contact with their neighbors and exhibited a fibroblast-like phenotype. The percentage of scattered colonies in total counted colonies (n>200) was measured. Values (means ± s.d.) are from three independent experiments. (F)MDCK cells stably expressing HA-PKCd were allowed to grow as colonies and then maintained in medium containing different amount of serum. Th...

show abstract

“…In untreated cells, PKC␦ was also found in the cytosol; however, a considerable fraction was associated with a juxtanuclear organelle, characterized earlier as the Golgi complex (21). The distinctive behavior of classical and novel PKC isoforms, despite their common ability to bind DAG, has been attributed to their differential ability to associate with other ligands such as arachidonic acid, ceramide, phosphatidylserine, or PKC-adaptor proteins including ␤-coatomer proteins (22)(23)(24). Remarkably, the vast majority of the novel kinase relocalized to the inclusion vacuole and/or its immediate vicinity when cells were infected by Chlamydia (Fig.…”

Section: Resultsmentioning

confidence: 99%

Accumulation of Diacylglycerol in the Chlamydia Inclusion Vacuole

Tse

Mason

Botelho

et al. 2005

Journal of Biological Chemistry

View full text Add to dashboard Cite

Intracellular pathogens have developed strategies to survive for extended periods inside their host cells. These include avoidance of host microbicidal effectors, often by sequestration in a protected subcompartment of the host cell. In some cases, the parasites exert also an antiapoptotic effect that prolongs the life of the infected host cell. Chlamydia utilizes both strategies, but the underlying molecular mechanisms are incompletely understood. Comparatively, little is known regarding the effects that Chlamydia exerts on the metabolism and distribution of the host cell lipids. The expression of fluorescently tagged C1 domains revealed that diacylglycerol is greatly accumulated in the immediate vicinity of Chlamydia inclusion vacuoles. The concentrated diacylglycerol recruits protein kinase C␦ (PKC␦), a proapoptotic effector, to the immediate vicinity of the vacuole. PKC␦ normally exerts its pro-apoptotic effects at the mitochondria and in the nucleus. We speculate that Chlamydia antagonizes the pro-apoptotic effect of PKC␦ by sequestering the enzyme on the inclusion vacuole away from its conventional target sites. Accordingly, we found that the ectopic expression of a catalytic fragment of PKC␦ that cannot be recruited by the vacuole, because it lacks a functional C1 domain, overcame the anti-apoptotic effect of the bacteria. The scavenging of pro-apoptotic factors may provide a novel mechanism whereby pathogens promote their own survival by extending the life of the host cells they infect.

show abstract

Identification of an Amino Acid Residue in the Protein Kinase C C1b Domain Crucial for Its Localization to the Golgi Network

Cited by 43 publications

References 44 publications

Functional suppression of E-cadherin by protein kinase Cδ

Functional suppression of E-cadherin by protein kinase Cδ

Elevated expression of protein kinase Cδ induces cell scattering upon serum deprivation

Accumulation of Diacylglycerol in the Chlamydia Inclusion Vacuole

Contact Info

Product

Resources

About