Phosphorylation is required for PMA- and cell-cycle-induced degradation of protein kinase Cδ

Srivastava, Jyoti; Procyk, Katarzyna J.; Iturrioz, Xavier; Parker, Peter J.

doi:10.1042/bj20020737

Cited by 43 publications

(47 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The reasons for this difference are unclear but may relate to the finding that, unlike other isoforms of PKC, PKC requires PMA-induced hyperphosphorylation for down-regulation (Srivastava et al 2002). It is conceivable, therefore, that the enzyme may be less efficiently phosphorylated in HIT-T15 cells, leading to its retention during PMA exposure.…”

Section: Discussionmentioning

confidence: 69%

Evidence that protein kinase Cdelta is not required for palmitate-induced cytotoxicity in BRIN-BD11 beta-cells

Welters

Smith²,

Tadayyon³

et al. 2004

Journal of Molecular Endocrinology

View full text Add to dashboard Cite

Chronic exposure of pancreatic -cells to saturated fatty acids leads to loss of viability, an effect that has been implicated in the process of -cell 'lipotoxicity' associated with the progression of type 2 diabetes. The mechanisms involved are unknown but recent evidence has implicated the isoform of protein kinase C (PKC ) in mediating fatty acid toxicity. We have investigated this proposition in the clonal insulin-secreting cell line, BRIN-BD11. BRIN-BD11 cells were found to undergo apoptosis when exposed to palmitate and this response was attenuated by the purportedly selective inhibitor of PKC , rottlerin. However, activation of PKC with the phorbol ester, phorbol-12-myristate-13-acetate (PMA), failed to promote cell death and down-regulation of PKC did not prevent the cytotoxic effects of palmitate. Moreover, rottlerin remained effective as a blocker of the palmitate response in cells depleted of PKC . Since rottlerin can inhibit various other kinases in addition to PKC , a range of additional kinase inhibitors was also tested. Of these, only the putative Ca 2+ /calmodulin-dependent protein kinase II (CaM kinase II) inhibitor, KN-62, was found to inhibit palmitate-induced cell death. However, this effect was not reproduced by a more selective pseudo-substrate inhibitor of CaM kinase II. Therefore, the present results reveal that palmitate induces cell death in BRIN-BD11 cells and suggest that this may involve the activation of a rottlerin (and KN-62)-sensitive kinase. However, it is clear that PKC is not required for this response.

show abstract

Section: Discussionmentioning

confidence: 69%

Evidence that protein kinase Cdelta is not required for palmitate-induced cytotoxicity in BRIN-BD11 beta-cells

Welters

Smith²,

Tadayyon³

et al. 2004

Journal of Molecular Endocrinology

View full text Add to dashboard Cite

show abstract

“…Regardless of the mechanism, these studies suggest that the Transduction Laboratories anti-PKC-␦ antibody is particularly poorly suited to comparing levels of PKC-␦ expression in resting and agonist-activated samples or to examining the relationship between PKC-␦-T 505 phosphorylation and PKC-␦ trafficking and/or downregulation. In this context, it is noteworthy that studies with this reagent have been interpreted as evidence that PKC-␦-T 505 phosphorylation regulates the kinetics of PKC-␦ downregulation (10). Because the BD Transduction Laboratories anti-PKC-␦ artifactually perceives PKC-␦-T 505 (or a related) phosphorylation as a decline in total PKC-␦ protein (which is a cause for concern, given that these experiments were performed with the rat sequence), these studies (as well as others in which this reagent was used to track PKC-␦ protein expression) deserve to be revisited using different methodologies.…”

Section: Specificity Of Bd Transduction Laboratories Anti-pkc-␦mentioning

confidence: 99%

Immunoblotting PKC-δ: a cautionary note from the bench

Rybin¹,

Steinberg²

2006

American Journal of Physiology-Cell Physiology

View full text Add to dashboard Cite

Antibodies that specifically recognize signaling proteins (or individual phosphorylation events at specific residues in proteins of interest) have become important tools in the study of signaling pathways. However, the recognition properties of many commercially available antibodies have not been fully characterized. In the course of studies exploring PKC-δ phosphorylation mechanisms in cardiomyocytes, we have demonstrated that a BD Transduction Laboratories anti-PKC-δ MAb (generally viewed as an anti-PKC-δ protein antibody) recognizes PKC-δ in resting, but not in PMA-treated, cardiomyocytes. The observations that PKC-δ immunoreactivity is preserved when cultures are treated with PMA in the presence of a the PKC inhibitor GF-109203X and that PKC-δ immunoreactivity is restored by in vitro acid phosphatase treatment indicate that the epitope recognized by the BD Transduction Laboratories anti-PKC-δ MAb is masked by phosphorylation. The BD Transduction Laboratories MAb is poorly suited for studies that compare PKC-δ expression in resting and agonist-activated samples (or in studies of the relationship between PKC-δ phosphorylation and PKC-δ downregulation) because it artifactually displays PKC-δ phosphorylation as a decline in total PKC-δ protein. Other studies have shown that two anti-PKC-δ-pY311 antibodies (manufactured by Cell Signaling Technology, Beverly, MA, and BioSource International, Camarillo, CA, respectively) specifically recognize stimulus-induced changes in PKC-δ-Y311 phosphorylation on the endogenous PKC-δ enzyme, but the Cell Signaling Technology anti-PKC-δ-pY311 antibody provides a better measure of Y311 phosphorylation in overexpressed PKC-δ. Collectively, these studies have identified features of anti-PKC-δ antibodies that affect the interpretation of immunoblot analysis experiments. These findings related to PKC-δ may be symptomatic of a more pervasive feature of immunoblot analysis studies of phosphoproteins in general.

show abstract

“…The critical role of PKC in EV1 entry and infection was also confirmed in a third set of experiments ( Figure 6C), in which chronic treatment of cells with a phorbol ester (PMA, 6 h) was used to downregulate PKC expression. Chronic PMA treatment is known to induce the degradation of both classical and novel PKCs (Srivastava et al, 2002), and it is not specific for PKC␣. The effectiveness of the treatment was confirmed by Western blotting ( Figure 6C).…”

Section: Protein Kinase C Activity Is Essential For the Internalizatimentioning

confidence: 99%

Clustering Induces a Lateral Redistribution of α2β1 Integrin from Membrane Rafts to Caveolae and Subsequent Protein Kinase C-dependent Internalization

Upla

Marjomäki

Kankaanpää

et al. 2004

MBoC

151

186

View full text Add to dashboard Cite

Integrin ␣2␤1 mediates the binding of several epithelial and mesenchymal cell types to collagen. The composition of the surrounding plasma membrane, especially caveolin-1-and cholesterol-containing membrane structures called caveolae, may be important to integrin signaling. On cell surface ␣2␤1 integrin was located in the raft like membrane domain, rich in GPI-anchored proteins, rather than in caveolae. However, when antibodies were used to generate clusters of ␣2␤1 integrin, they started to move laterally on cell surface along actin filaments. During the lateral movement small clusters fused together. Finally ␣2␤1 integrin was found inside caveolae and subsequently internalized into caveosome-like perinuclear structures. The internalization process, unlike cluster formation or lateral redistribution, was dependent on protein kinase C␣ activity. Caveolae are known to be highly immobile structures and ␣2␤1 integrin clusters represent a previously unknown mechanism to activate endocytic trafficking via caveolae. The process was specific to ␣2␤1 integrin, because the antibody-mediated formation of ␣V integrin clusters activated their internalization in coated vesicles and early endosomes. In addition to natural ligands human echovirus-1 (EV1) gains entry into the cell by binding to ␣2␤1 and taking advantage of ␣2␤1 internalization via caveolae. INTRODUCTIONCaveolae are cave-like invaginations of the cell surface (for reviews see Kurzchalia and Parton, 1999;Parton 2003). They have been described as highly immobile and not involved in constitutive endocytic trafficking (Thomsen et al., 2002). Their internalization can be activated, for example, by the phosphatase inhibitor okadaic acid (Parton et al., 1994). In endothelial cells the interaction of albumin docking protein gp60 and caveolin-1 initiates the endocytosis of caveolae (Minshall et al., 2000). Caveolins are membrane proteins that are molecular markers of caveolae and shown to be crucial for the formation of these structures (Fra et al., 1995). Caveolins can be associated with cell surface growth factor receptors (Couet et al., 1997) and cell adhesion receptors (Wary et al., 1996(Wary et al., , 1998Wei et al., 1999) and caveolae might therefore play a role in cellular signaling. In addition, caveolae have been suggested to participate in the uptake of folate by pinocytosis, but this is still under discussion (Parton, 2003). Caveolin-deficient mice have given novel insight into the function of caveolae. Caveolin-1 gene knockout leads to pulmonary defects, vasoconstriction, or dilatation abnormalities and resistance to diet-induced obesity (Drab et al., 2001;Razani et al., 2001;Schubert et al., 2001;Sotgia et al., 2002;Razani et al., 2002). Simian virus 40 (SV40) has been shown to be internalized through caveolae and detailed studies focused on the virus entry mechanism have produced a lot of new information about the biology of caveolae (Pelkmans et al., 2001. We have recently shown that human echovirus 1 (EV1) is another virus using caveolae in its entry (Mar...

show abstract

Phosphorylation is required for PMA- and cell-cycle-induced degradation of protein kinase Cδ

Cited by 43 publications

References 35 publications

Evidence that protein kinase Cdelta is not required for palmitate-induced cytotoxicity in BRIN-BD11 beta-cells

Evidence that protein kinase Cdelta is not required for palmitate-induced cytotoxicity in BRIN-BD11 beta-cells

Immunoblotting PKC-δ: a cautionary note from the bench

Clustering Induces a Lateral Redistribution of α2β1 Integrin from Membrane Rafts to Caveolae and Subsequent Protein Kinase C-dependent Internalization

Contact Info

Product

Resources

About