Ligand Structure-Activity Requirements and Phospholipid Dependence for the Binding of Phorbol Esters to Protein Kinase D

Abstract

Although protein kinase D (PKD), like protein kinase C (PKC), possesses a C1 domain that binds phorbol esters and diacylglycerol, the structural differences from PKC within this and other domains of PKD imply differential regulation by lipids and ligands. We characterized the phorbol ester and phospholipid binding properties of a glutathione S-transferase-tagged fulllength PKD and compared them with those of PKC-␣ and -␦. We found that PKD is a high-affinity phorbol ester receptor for a range of structurally and functionally divergent phorbol esters and analogs and showed both similarities and differences in structure-activity relations compared with the PKCs examined. In particular, PKD had lower affinity than PKC for certain diacylglycerol analogs, which might be caused by a lysine residue at the 22 position of the PKD-C1b domain in place of the tryptophan residue at this position conserved in the PKCs. The membrane-targeting domains in PKD are largely different from those in PKC; among these differences, PKD contains a pleckstrin homology (PH) domain that is absent in PKC. However, phosphatidylinositol-4,5-bisphosphate PIP 2 , a lipid ligand for some PH domains, reconstitutes phorbol 12,13-dibutyrate (PDBu) binding to PKD similarly as it does to PKC-␣ and -␦, implying that the PH domain in PKD may not preferentially interact with PIP 2 . Overall, the requirement of anionic phospholipids for the reconstitution of [ 3 H]PDBu binding to PKD was intermediate between those of PKC-␣ and -␦. We conclude that PKD is a high-affinity phorbol ester receptor; its lipid requirements for ligand binding are approximately comparable with those of PKC but may be differentially regulated in cells through the binding of diacylglycerol to the C1 domain.

Diacylglycerol-dependent Binding Recruits PKCθ and RasGRP1 C1 Domains to Specific Subcellular Localizations in Living T Lymphocytes

“…DOG might insert more easily into the binding pocket than SAG, explaining C1RasGRP1 differential specificity for inner or plasma membrane DAG. Similar observations have been made for the DAG analog specificity of other C1-containing proteins (Wang et al, 2003). Binding groove differences may also explain C1 domain conformational flexibility, which may account for ligand affinity and membrane penetration (Ananthanarayanan et al, 2003) RasGRP1 translocation to Golgi in response to soluble anti-CD3 and -CD28 was recently described , although here we show clear localization to the site in contact with anti-CD3ϩCD28 -coated microspheres.…”

Role of the Regulatory Domain of Protein Kinase D2 in Phorbol Ester Binding, Catalytic Activity, and Nucleocytoplasmic Shuttling

“…Like the other PKD family members, the kinase contains a regulatory domain at the amino terminus comprising a duplex cysteine-rich domain, C1a/C1b, and a PH domain. Regulatory domains determine the basal activity of protein kinases and contribute to the regulation of their subcellular localization (Oancea et al, 2003;Rykx et al, 2003;Wang et al, 2003). The aim of our study was to determine whether the regulatory domain in PKD2 has similar or distinct effects on the regulation of PDBu binding, catalytic activity and subcellular localization of the kinase.…”

“…Cysteine-rich zinc finger-like motifs are present in a variety of proteins, including PKCs (Wang et al, 2003), chimaerin (Areces et al, 1994), and UNC-13 (Kazanietz et al, 1995), and they play multiple roles in enzyme regulation. In PKD1, the C1a/C1b domain is responsible for the majority of phorbol ester binding and acts as a membrane targeting module that shuttles PKD1 between different subcellular compartments (Rykx et al, 2003).…”

Ceramide signaling in cancer and stem cells

“…However, there have been successful studies to determine binding proteins and even specific binding domains for many other signaling lipids. For example, phosphatidylinositolphosphates (PIPs) bind to the pleckstrin homology (PH) domain and structurally related domains [30,31]. Diacylglycerol (DAG), an important pro-proliferative lipid, interacts with the C1 domain of classical PKC (PKCα) and PKD [31][32][33][34][35].…”

“…For example, phosphatidylinositolphosphates (PIPs) bind to the pleckstrin homology (PH) domain and structurally related domains [30,31]. Diacylglycerol (DAG), an important pro-proliferative lipid, interacts with the C1 domain of classical PKC (PKCα) and PKD [31][32][33][34][35]. Within the family of sphingolipids, binding partners have only been specified for ceramide [29,[36][37][38][39][40][41][42][43][44][45][46], ceramide-1-phosphate [47][48][49], sphingosine-1-phosphate (S1P) [19,[50][51][52] and some gangliosides (e.g., GM1).…”