Calcium stimulates self-assembly of protein kinase C α in vitro

Carter J. Swanson, Ruth Sommese, Karl J. Petersen, Michael Ritt, Joshua Karslake, David D. Thomas, Sivaraj Sivaramakrishnan

Research output: Contribution to journalArticlepeer-review

8 Scopus citations

Abstract

Protein kinase C a (PKCα) is a nodal regulator in several intracellular signaling networks. PKCα is composed of modular domains that interact with each other to dynamically regulate spatial-temporal function. We find that PKCα specifically, rapidly and reversibly selfassembles in the presence of calcium in vitro. This phenomenon is dependent on, and can be modulated by an intramolecular interaction between the C1a and C2 protein domains of PKCα. Next, we monitor self-assembly of PKCmCitrine fusion proteins using timeresolved and steady-state homoFRET. HomoFRET between full-length PKCα molecules is observed when in solution with both calcium and liposomes containing either diacylglycerol (DAG) or phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). Surprisingly, the C2 domain is sufficient to cluster on liposomes containing PI(4,5)P2, indicating the C1a domain is not required for self-assembly in this context. We conclude that three distinct clustered states of PKCα can be formed depending on what combination of cofactors are bound, but Ca2+ is minimally required and sufficient for clustering.

Original languageEnglish (US)
Article numbere0162331
JournalPloS one
Volume11
Issue number10
DOIs
StatePublished - Oct 2016

Bibliographical note

Funding Information:
DLS experiments were performed using the Biophysics Program common research facilities at the University of Michigan with acknowledgment to Samuel Kotler and A. Rams Ramamoorthy for technical advice and assistance with the instrument. Thomas van Zanten and Satyajit Mayor provided valuable feedback on the manuscript preparation and performed preliminary time-resolved fluorescence experiments. The reported time-resolved fluorescence measurements were performed in the Biophysical Technology Center, University of Minnesota, with support from an NIH Small Business Technology Transfer grant (R42 DA037622). This study was supported by the American Heart Association Scientist Development Grant (13SDG14270009) & the NIH (1DP2 CA186752-01 & 1-R01-GM-105646-01-A1) to S.S. K.J.P. was supported by an NIH training grant (T32 AR007612). R.F.S. is a Life Sciences Research Foundation postdoctoral fellow.

Publisher Copyright:
© 2016 Swanson et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Fingerprint

Dive into the research topics of 'Calcium stimulates self-assembly of protein kinase C α in vitro'. Together they form a unique fingerprint.

Cite this