Abstract 2373: Analysis of the distinct activities of the C1a and C1b domains of protein kinase C theta

• Published in: Cancer research (Chicago, Ill.) Vol. 77; no. 13_Supplement; p. 2373
• Main Authors: Czikora, Agnes, Pany, Satyabrata, You, Youngki, Saini, Amandeep, Kedei, Noemi, Lewin, Nancy E., Abramovitz, Adelle, Das, Joydip, Blumberg, Peter M.
• Format: Journal Article
• Language: English
• Published: 01-07-2017
• ISSN: 0008-5472; 1538-7445
• DOI: 10.1158/1538-7445.AM2017-2373

PKC isozymes, the major receptors for tumor-promoting phorbol esters, play a central role in cellular signal transduction downstream of the second messenger diacylglycerol. The PKCθ isozyme has been extensively studied particularly in the context of hematopoiesis and immunity, where is required for the development and maturation of specific T cell subsets. PKCθ also plays a role in Notch-driven T cell leukemia and in a subset of gastrointestinal stromal tumors, melanoma and breast cancers. The C1 domains represent the regulatory motif in the PKCs responsible for their recognition of diacylglycerol / phorbol esters. PKCθ is unique among the conventional and novel PKC isoforms in that one of the two C1 domains of PKCθ, the C1a domain, has been reported to show little or no affinity for diacylglycerol or phorbol ester, unlike the C1b domain. Likewise, the PKCθ C1b domain has been reported to play the predominant role in the membrane translocation and activation process of PKCθ. We observed that the C1a domain in fact has appreciable binding activity for [3H]phorbol 12,13-dibutyrate (PDBu), with a Kd of 254 nM, albeit much less than that of the C1b domain, 1.58 nM. Replacing individually in the C1a domain ten of the amino acid residues that differed between the C1a and C1b domains, using the corresponding residue from the C1b domain, we found that only the P9K replacement restored [3H]phorbol 12,13-dibutyrate binding activity to the C1a domain, yielding a Kd of 3.60 nM, close to that of the C1b domain. Strikingly, the reciprocal replacement in the C1b domain of K9P still preserved appreciable binding affinity, 11.7 nM. We conclude that other features in the C1a domain contribute to the extent to which the P9 residue negatively influences binding. We further explored the role of the C1 domains of PKCθ on translocation using confocal microscopy with a variety of full length constructs in which the C1 domains were manipulated. Consistent with the binding studies, we showed that the C1b domain was the main contributor to translocation, that the full length PKCθ with the C1b K9P retained response, albeit more weakly, and that deletion of the C1a domain or its replacement with a second C1b domain enhanced the translocation to the plasma membrane. Exploitation of the structural differences between the C1 domains in PKCθ and those of other PKC isoforms, both at the level of the direct ligand interactions and the more complicated interplay between C1 domain, ligand, and phospholipid bilayer, may help to afford specific ligands for this member of the PKC family.
Citation Format: Agnes Czikora, Satyabrata Pany, Youngki You, Amandeep Saini, Noemi Kedei, Nancy E. Lewin, Adelle Abramovitz, Joydip Das, Peter M. Blumberg. Analysis of the distinct activities of the C1a and C1b domains of protein kinase C theta [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2373. doi:10.1158/1538-7445.AM2017-2373