Novel phosphorylation site markers of protein kinase C delta activation

Durgan, Joanne; Michael, Nicholas; Totty, Nick; Parker, Peter J.

doi:10.1016/j.febslet.2007.06.035

Cited by 33 publications

(47 citation statements)

References 24 publications

(27 reference statements)

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“…Several residues located in the hinge region of PKCδ, including Ser299, Ser302, Ser304, Tyr311 and Tyr332, have been shown to be phosphorylated under certain circumstances (Konishi et al, 2001;Durgan et al, 2007). Some of Journal of Cell Science 122 (4) these phosphorylation events are already known to regulate the catalytic activity of PKCδ (Konishi et al, 2001;Durgan et al, 2007). However, it is not clear whether any of these phosphorylation events could modulate the targeting of PKCδ to cell-cell junctions.…”

Section: Discussionmentioning

confidence: 98%

“…521 Functional suppression of E-cadherin by PKCδ these phosphorylation events are already known to regulate the catalytic activity of PKCδ (Konishi et al, 2001;Durgan et al, 2007). However, it is not clear whether any of these phosphorylation events could modulate the targeting of PKCδ to cell-cell junctions.…”

Section: Journal Of Cell Sciencementioning

confidence: 99%

“…4A), we speculate that a stretch of 19 amino acids (280-298) of PKCδ might be the most critical determinant for its targeting of cell-cell junctions. Several residues located in the hinge region of PKCδ, including Ser299, Ser302, Ser304, Tyr311 and Tyr332, have been shown to be phosphorylated under certain circumstances (Konishi et al, 2001;Durgan et al, 2007). Some of Journal of Cell Science 122 (4) …”

Section: Journal Of Cell Sciencementioning

confidence: 99%

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Functional suppression of E-cadherin by protein kinase Cδ

Chen

2009

Journal of Cell Science

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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...

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Section: Discussionmentioning

confidence: 98%

Section: Journal Of Cell Sciencementioning

confidence: 99%

Section: Journal Of Cell Sciencementioning

confidence: 99%

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Functional suppression of E-cadherin by protein kinase Cδ

Chen

2009

Journal of Cell Science

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“…25 mapped to the pseudosubstrate sequence (Thr-141) and to three sites in the hinge domain (Ser-295/Ser-299/Ser-304) on PKC [96,123] (Figure 3). Phosphorylation of PKC at Ser-299 serves as a useful marker for catalytic activation of the enzyme [96].…”

Section: Accepted M Manuscriptmentioning

confidence: 99%

“…Phosphorylation of PKC at Ser-299 serves as a useful marker for catalytic activation of the enzyme [96].…”

Section: Accepted M Manuscriptmentioning

confidence: 99%

Regulation of Protein Kinase C function by phosphorylation on conserved and non-conserved sites

Freeley

Kelleher

Long

2011

Cellular Signalling

105

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Protein kinase C‐δ interacts with and phosphorylates ARD1

Chun

Cho

Lee

et al. 2020

Journal Cellular Physiology

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Protein kinase C-δ (PKCδ) is a diacylglycerol-dependent, calcium-independent novel PKC isoform that is engaged in various cell signaling pathways, such as cell proliferation, apoptosis, inflammation, and oxidative stress. In this study, we searched for proteins that bind PKCδ using a yeast two-hybrid assay and identified murine arrest-defective 1 (mARD1) as a binding partner. The interaction between PKCδ and mARD1 was confirmed by glutathione S-transferase pull-down and coimmunoprecipitation assays. Furthermore, recombinant PKCδ phosphorylated fulllength mARD1 protein. The NetPhos online prediction tool suggested PKCδ phosphorylates Ser 80 , Ser 108 , and Ser 114 residues of mARD1 with the highest probability. Based on these results, we synthesized peptides containing these sites and examined their phosphorylations using recombinant PKCδ. Autoradiography confirmed these sites were efficiently phosphorylated. Consequent mass spectrometry and peptide sequencing in combination with MALDI-TOF MS/MS confirmed that Ser 80 and Ser 108 were major phosphorylation sites. The alanine mutations of Ser 80 and Ser 108 abolished the phosphorylation of mARD1 by PKCδ in 293T cells supporting these observations. In addition, kinase assays using various PKC isotypes showed that Ser 80 of ARD1 was phosphorylated by PKCβI and PKCζ isotypes with the highest selectivity, while Ser 108 and/or Ser 114 were phosphorylated by PKCγ with activities comparable to that of the PKCδ isoform. Overall, these results suggest the possibility that PKCδ transduces signals by regulating phosphorylation of ARD1.

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Novel phosphorylation site markers of protein kinase C delta activation

Cited by 33 publications

References 24 publications

Functional suppression of E-cadherin by protein kinase Cδ

Functional suppression of E-cadherin by protein kinase Cδ

Regulation of Protein Kinase C function by phosphorylation on conserved and non-conserved sites

Protein kinase C‐δ interacts with and phosphorylates ARD1

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