Protein Kinase C-Delta (PKCδ) Tyrosine Phosphorylation is a Critical Regulator of Neutrophil-Endothelial Cell Interaction in Inflammation

Abstract Abstract 3004 Poster Board II-981 Protein Kinase C-delta (PKCδ) is a novel PKC isoform that differentially regulates platelet dense granule secretion. PKCδ positively regulates Protease activated receptor (PAR)-mediated dense granule secretion, whereas it negatively regulates glycoproteinVI (GPVI)-mediated dense granule secretion in platelets. PKCδ, a serine/threonine kinase is phosphorylated on its tyrosine residues. There are nine potential tyrosine phosphorylation sites in the regulatory domain of PKCδ. Phosphorylation at different tyrosine residues regulates its substrate specificity. We have previously shown that the association of PKCδ with Lyn and SHIP-1 negatively regulates GPVI-mediated dense granule secretion. However, the event leading to the association between PKCδ and SHIP-1 is not known. We hypothesize that the differential tyrosine phosphorylation of PKCδ downstream of PARs or GPVI receptors result in the preferential association with SHIP-1. In the current study, we show that PKCδ is phosphorylated at tyrosine residues Y332, Y523, Y525 and Y565 upon PAR or GPVI stimulation. Y311 residue is predominantly phosphorylated upon stimulation of PARs, whereas Y155 residue is preferentially phosphorylated upon GPVI stimulation. PAR-mediated Y311 phosphorylation peaks at later timepoint, whereas GPVI-mediated Y155 phosphorylation peaks at an early timepoint. correlating with dense granule secretion. Furthermore, we show that agarose-conjugated Y155 phosphorylated PKCδ peptide associates with SHIP-1 upon GPVI stimulation, and not PARs. These data suggest that the phosphorylation of PKCδ at distinct tyrosine residues differentially regulate its association with SHIP-1. Therefore, we conclude that the GPVI-mediated phosphorylation of PKCδ at 155 is required for its association with SHIP-1. This study is supported by pre-doctoral fellowship to Ramya Chari from American Heart Association, Pennsylvania-Delaware affiliate. Disclosures: No relevant conflicts of interest to declare.

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Expression of an oncogenic rasHa gene in murine keratinocytes induces tyrosine phosphorylation and reduced activity of protein kinase C delta.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)74282-3 ◽

1993 ◽

Vol 268 (35) ◽

pp. 26079-26081

Author(s):

M F Denning ◽

A A Dlugosz ◽

M K Howett ◽

S H Yuspa

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Tyrosine Phosphorylation ◽

Protein Kinase C Delta ◽

Kinase C ◽

Reduced Activity

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Tyrosine phosphorylation of protein kinase C-delta in response to the activation of the high-affinity receptor for immunoglobulin E modifies its substrate recognition.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.92.20.9112 ◽

1995 ◽

Vol 92 (20) ◽

pp. 9112-9116 ◽

Cited By ~ 62

Author(s):

H. Haleem-Smith ◽

E. Y. Chang ◽

Z. Szallasi ◽

P. M. Blumberg ◽

J. Rivera

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Tyrosine Phosphorylation ◽

Immunoglobulin E ◽

Substrate Recognition ◽

Protein Kinase C Delta ◽

High Affinity ◽

Kinase C ◽

High Affinity Receptor ◽

Affinity Receptor

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Regulation of lysophosphatidic acid-stimulated tyrosine phosphorylation of mitogen-activated protein kinase by protein kinase C- and pertussis toxin-dependent pathways in the endothelial cell line EAhy 926

Biochemical Journal ◽

10.1042/bj3070743 ◽

1995 ◽

Vol 307 (3) ◽

pp. 743-748 ◽

Cited By ~ 17

Author(s):

A McLees ◽

A Graham ◽

K Malarkey ◽

G W Gould ◽

R Plevin

Keyword(s):

Protein Kinase C ◽

Endothelial Cell ◽

Protein Kinase ◽

Map Kinase ◽

Cell Line ◽

Tyrosine Phosphorylation ◽

Pertussis Toxin ◽

Lysophosphatidic Acid ◽

Kinase C ◽

Mitogen Activated Protein

In the endothelial cell line EAhy 926, 1-oleoyl-lysophosphatidic acid (LPA) stimulated the tyrosine phosphorylation of the pp42 isoform of mitogen-activated protein (MAP) kinase. Maximum phosphorylation was observed within 5 min of LPA addition, but the response was sustained for up to 120 min. Re-addition of LPA after 60 min stimulated a further sustained increase in the tyrosine phosphorylation of MAP kinase. In cells pretreated with phorbol 12-myristate 13-acetate (PMA; 24 h) or preincubated with the protein kinase C inhibitor Ro-318220, LPA-induced tyrosine phosphorylation of pp42 MAP kinase was substantially reduced at 2 min but potentiated at 60 min. Ro-318220 in combination with either PMA or pertussis toxin pretreatment abolished the LPA response at all time points, suggesting an involvement of protein kinase C in the pertussis toxin-sensitive part of the pathway. Agents which raised intracellular cyclic AMP levels did not affect the initial phase of LPA-stimulated MAP kinase activation, but abolished the late phase. However, this effect was prevented by Ro-318220, implicating a greater role for protein kinase C than protein kinase A in the regulation of sustained MAP kinase responses. LPA stimulated an increase in the tyrosine phosphorylation of focal adhesion kinase pp125 (pp125FAK) in EAhy 926 cells which was both protein kinase C- and pertussis toxin-independent. These results are discussed in terms of the pathways regulating both MAP kinase and pp125FAK in response to LPA in the EAhy 926 endothelial cells line.

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