AbstractThe FAM83 family of proteins are classified based on the presence of a conserved domain of unknown function 1669 (DUF1669) within their N-termini. Although structural and bioinformatic studies suggest that the DUF1669 domain adopts a phospholipase D-like motif, no phospholipase D activity has yet been demonstrated for FAM83 members experimentally. Thus, the role and function of FAM83 proteins remains elusive. This study provides evidence that FAM83 proteins bind to different isoforms of the casein kinase 1 (CK1) family of Ser/Thr protein kinases and direct them to the distinct cellular sites in which the FAM83 proteins reside. FAM83 members mediate CK1 binding through their conserved DUF1669. In this capacity, FAM83 proteins may act as the A Kinase Anchoring proteins (AKAPs) of the CK1 world, and act analogously to the way AKAPs serve to regulate protein kinase A.
Regarded as constitutively-active, promiscuous protein kinases, the regulation of CK1 isoforms is critically important, yet poorly understood. This is particularly crucial when considering the reported participation of CK1 kinases in many, diverse signalling processes, from Wnt signalling to the regulation of Circadian rhythm. Mechanisms regulating CK1 isoforms, through controlling their cellular localisation and therefore substrate accessibility, are thus attractive regulatory targets to facilitate the study into specific CK1 functions, which rely on the recruitment of CK1 isoforms to the relevant cellular sites. As FAM83 proteins have the potential to act in such a manner, investigation was focussed on specific FAM83-CK1 interactions in order to further define this emerging regulatory role of FAM83 members.
FAM83D is unique amongst the FAM83 proteins, in that it is the only FAM83 member known to localise to the mitotic spindle. RNA interference approaches targeting FAM83D have been reported to impact chromosome alignment and cause a delay in the timings of cell division. Whilst all FAM83 proteins appear to associate robustly with the CK1 alpha isoforms (CK1α), interestingly, the interaction between FAM83D and CK1α in asynchronous cell extracts was weak. Armed with the knowledge that FAM83D localises to the spindle apparatus in mitosis, a hypothesis that the FAM83D-CK1α interaction might only occur during cell division was developed. This study provides evidence that FAM83D binds and recruits CK1α to the mitotic spindle, and that the FAM83D-CK1α interaction is critical for correct and efficient spindle positioning. Cells devoid of FAM83D, or those harbouring a FAM83D CK1-binding- deficient knockin point mutation, fail to localise CK1α to mitotic spindles, and present with spindle orientation defects, and a concurrent delay in the metaphase-to-anaphase transition. As the spindle position determines the axis of cell division, correctly- orientated spindle positioning is critically important in both development and in the maintenance of healthy adult tissues.
When one thinks of mitotic kinases regulating spindle positioning, prominent players such as cyclin-dependent kinases, aurora kinases, and polo-like kinases tend to dominate the cell division limelight. However, these kinase families alone cannot account for the extent of protein phosphorylation reported over a cell division cycle, implying roles for other protein kinases in this process. Thus, implicating CK1α as a mitotic kinase provides further mechanistic insights into the regulation of mitosis, and the phosphorylation-mediated control of cell division.
As FAM83 proteins are an emerging family of proteins, tools to study them are scarce, yet warranted. In this vein, part of this study was devoted to developing unique approaches and technologies to aid in the investigation of FAM83 biology, and beyond. In this context, two nanobody-based strategies to either promote the phosphorylation of the target protein, or mark it for proteasomal degradation are described. These novel tools have been employed here to explore the role of the FAM83D-CK1α interaction in cells, and I provide evidence that they are applicable for research into diverse biological processes and questions.
|Date of Award||2019|
|Supervisor||Gopal Sapkota (Supervisor) & Ian Ganley (Supervisor)|
- cell division
- protein phosphorylation
- cell biology