The DUF1669 domain of FAM83 family proteins anchor casein kinase 1 isoforms

Abstract

Members of the casein kinase 1 (CK1) family of serine-threonine protein kinases are implicated in the regulation of many cellular processes, including the cell cycle, circadian rhythms, and Wnt and Hedgehog signaling. Because these kinases exhibit constitutive activity in biochemical assays, it is likely that their activity in cells is controlled by subcellular localization, interactions with inhibitory proteins, targeted degradation, or combinations of these mechanisms. We identified members of the FAM83 family of proteins as partners of CK1 in cells. All eight members of the FAM83 family (FAM83A to FAM83H) interacted with the α and α-like isoforms of CK1; FAM83A, FAM83B, FAM83E, and FAM83H also interacted with the δ and ε isoforms of CK1. We detected no interaction between any FAM83 member and the related CK1γ1, CK1γ2, and CK1γ3 isoforms. Each FAM83 protein exhibited a distinct pattern of subcellular distribution and colocalized with the CK1 isoform(s) to which it bound. The interaction of FAM83 proteins with CK1 isoforms was mediated by the conserved domain of unknown function 1669 (DUF1669) that characterizes the FAM83 family. Mutations in FAM83 proteins that prevented them from binding to CK1 interfered with the proper subcellular localization and cellular functions of both the FAM83 proteins and their CK1 binding partners. On the basis of its function, we propose that DUF1669 be renamed the polypeptide anchor of CK1 domain.

FAM83D directs protein kinase CK1α to the mitotic spindle for proper spindle positioning

“…Although FAM83D (aka CHICA) is poorly characterised, it has been shown to be recruited to the mitotic spindle through its association with the microtubule-associated protein hyaluronan mediated motility receptor (HMMR, aka RHAMM or CD168) (Connell, Chen et al, 2017, Dunsch, Hammond et al, 2012, Santamaria, Nagel et al, 2008. Unlike the other FAM83 members that appear to associate robustly with CK1a, we found that over-expressed GFP-FAM83D in asynchronous cell extracts interacted rather weakly, yet selectively, with CK1α (Fulcher et al, 2018), suggesting this could be a regulated interaction. Consistent with this, in the course of an unbiased proteomic approach to identify interactors of endogenous FAM83D from both asynchronous and mitotic extracts, we discovered in this study that FAM83D interacts with CK1a only in mitosis.…”

“…Mitotic cells were collected by shake-off, following either prometaphase arrest with nocodazole and a brief release into fresh medium to allow them to progress into mitosis, or mitotic arrest with the Eg5 chromokinesin inhibitor S-trityl L-cysteine (STLC), which results in monopolar spindle formation (Skoufias, DeBonis et al, 2006). Mass spectrometric analysis of anti-GFP IPs from both asynchronous and mitotic cell extracts identified several known FAM83D interactors, including HMMR, DYNLL1, and the transcription factor BACH1 (Dunsch et al, 2012, Fulcher et al, 2018, Li, Shiraki et al, 2012 (Fig. 1B), potentially revealing the constitutive FAM83D interactors.…”

“…Regarded as constitutively-active protein kinases, the regulation of CK1 isoforms is critically important, yet poorly understood; especially when considering their participation in multiple, diverse, cellular functions in many, different cellular compartments (Knippschild et al, 2005, Schittek & Sinnberg, 2014. We recently reported that the FAM83 family of poorly characterised proteins act as subcellular anchors for CK1 isoforms through the conserved N-terminal domain of unknown function 1669 (DUF1669) (Fulcher, Bozatzi et al, 2018). Our findings that FAM83 proteins interact and co-localise with different CK1 isoforms offer the tantalising possibility that FAM83 proteins direct CK1 isoforms to specific subcellular compartments, and in doing so, regulate their substrate availability/accessibility.…”

FAM 83D directs protein kinase CK 1α to the mitotic spindle for proper spindle positioning

“…Although FAM83D (aka spindle protein CHICA) is poorly characterised, it has been shown to be recruited to the mitotic spindle through its association with the microtubule‐associated protein hyaluronan‐mediated motility receptor (HMMR, aka RHAMM or CD168) . Unlike the other FAM83 members that appear to associate robustly with CK1α, we found that over‐expressed green fluorescent protein (GFP)‐tagged FAM83D in asynchronous cell extracts interacted rather weakly, yet selectively, with CK1α , suggesting this could be a regulated interaction. Consistent with this, in the course of an unbiased proteomic approach to identify interactors of endogenous FAM83D from both asynchronous and mitotic extracts, we discovered in this study that FAM83D interacts with CK1α only in mitosis.…”

“…HMMR, which is responsible for recruiting FAM83D to the spindle in mitosis [16], was observed at the spindle in wild-type, FAM83D À/À and FAM83D GFP/GFP cells ( Fig EV2D). We previously identified two conserved residues (equivalent to D 249 and F 283 of FAM83D) within the conserved DUF1669 of FAM83 proteins that were critical for mediating the FAM83-CK1 interaction [12]. GFP-tagged wild-type FAM83D, but not F283A or D249A mutants, co-precipitated endogenous CK1a during mitosis when transiently expressed in FAM83D À/À cells ( Fig EV3A).…”