1H, 13C and 15N NMR assignments of the C1A and C1B subdomains of PKC-delta

Abstract

The Protein Kinase C family of enzymes is a group of serine/threonine kinases that play central roles in cell-cycle regulation, development and cancer. A key step in the activation of PKC is translocation to membranes and binding of membrane-associated activators including diacylglycerol (DAG). Interaction of novel and conventional isotypes of PKC with DAG and phorbol esters occurs through the two C1 regulatory domains (C1A and C1B), which exhibit distinct ligand binding selectivity that likely controls enzyme activation by different co-activators. PKC has also been implicated in physiological responses to alcohol consumption and it has been proposed that PKCα (Slater et al. J Biol Chem 272(10):6167-6173, 1997; Slater et al. Biochemistry 43(23):7601-7609, 2004), PKCε (Das et al. Biochem J 421(3):405-413, 2009) and PKCδ (Das et al. J Biol Chem 279(36):37964-37972, 2004; Das et al. Protein Sci 15(9):2107-2119, 2006) contain specific alcohol-binding sites in their C1 domains. We are interested in understanding how ethanol affects signal transduction processes through its affects on the structure and function of the C1 domains of PKC. Here we present the (1)H, (15)N and (13)C NMR chemical shift assignments for the Rattus norvegicus PKCδ C1A and C1B proteins.

Figure 1

Alignment of protein constructs used in this study. Amino acids derived from the wild-type C1A and C1B domains are in black and residues derived from the pGEX-4T1 vector are in grey. The extents of the conserved C1 domains are indicated in the boxed region, identical residues are indicated with an asterisk (*) and strongly conserved residues are indicated with a colon (:). Alignment and analysis based on CLUSTAL W algorithm [21].

Figure 2

¹H,¹⁵N-HSQC spectrum of PKCδ C1A recorded at 600 MHz. Each peak labeled with the chemical shift assignments. The side chain amides for Asn and Gln residues are linked by horizontal bars.

Figure 3

¹H,¹⁵N-HSQC spectrum of PKCδ C1B recorded at 600 MHz. Each observable amide is labeled with its assignments within this construct. The side chain amides for Asn and Gln residues are linked by horizontal bars.

Figure 4

Ribbon diagram of the core C1B domain of PKCδ (based on PDB ID 1PTQ [12]). The location of the two zinc binding sites are shown in orange. Residues that cannot be observed in either ¹H,¹⁵N-HSQC or ¹H,¹³C-HSQC are colored red. This includes almost all residues in the phorbol acetate binding loops between residues N237-T242 and L251-G258. This directly contrast with the C1A domain, where all these residues can be observed