Kinase regulation by liquid-liquid phase separation

Abstract

Liquid-liquid phase separation (LLPS) is emerging as a mechanism of spatiotemporal regulation that could answer long-standing questions about how order is achieved in biochemical signaling. In this review we discuss how LLPS orchestrates kinase signaling, either by creating condensate structures that are sensed by kinases or by direct LLPS of kinases, cofactors, and substrates - thereby acting as a mechanism to compartmentalize kinase-substrate relationships, and in some cases also sequestering the kinase away from inhibitory factors. We also examine the possibility that selective pressure promotes genomic rearrangements that fuse pro-growth kinases to LLPS-prone protein sequences, which in turn drives aberrant kinase activation through LLPS.

Keywords: intrinsically disordered region; kinase; kinase fusion; liquid–liquid phase separation; multivalency; post-translational modification.

Figure 1.. Condensates as organizing platforms for kinase signaling.

A) Some kinases are recruited to ubiquitin-rich condensates either directly through ubiquitin-interacting domains (UBDs), or indirectly through protein-protein interactions with ubiquitin-interacting proteins (adaptors). TNK1 has a C-terminal UBA domain which binds poly-ubiquitin with high affinity. TBK1 is a Ser/Thr kinase that interacts with adaptor proteins such as NDP52, OPTN and SINTBAD, which possess UBDs in their C-terminal domains (CTDs). At condensates, TBK1-mediated phosphorylation of p62 and OPTN increases their affinity to ubiquitin, serving as a feedforward mechanism to promote condensate growth. B) Some kinases can be recruited to SGs either by protein-protein interactions with SG-associated proteins or via their IDRs. For instance, G3BP1 (represented with a blue circle bound to RNPs), a SG-associated protein, recruits protein kinase R to SG to become active. In addition, the yeast kinase Sky1 is recruited to SGs via its prion-like domain (PrLD), where it can phosphorylate substrates, such as Npl3 (represented with a magenta circle bound to RNPs) to enhance SG disassembly. Therefore, Sky1 recruitment and activation at SGs serves as negative feedback for SG growth. Other RNP bidning proteins are represented with green circles bound to RNPs. Stress granule cores are represented in gray inside phase separated condensates (white) that form, together, a mature biphasic stress granule as explained in [180]. This image was created with Biorender.com

Figure 2.. LLPS as a mode of kinase regulation.

A) In the ring-like state, Polo-like kinase 4 (Plk4) is mostly inactive and interacts with Cep152 (not shown) at centrioles. While a phosphorylation event within its L1 domain inhibits its ability to phase separate, autophosphorylation at the CPB PC3 motif causes a nearby region called the PB2 tip to become disordered, inducing LLPS of Plk4. In this phase separated state, Plk4 is mostly active. Plk4 LLPS facilitates the recruitment and phosphorylation of its substrate STIL, and the recruitment of Sas6 to promote centriole biogenesis. B) cAMP binding to the regulatory subunit RIα promotes the dissociation of the catalytic subunits of PKA and causes the inhibitory sequence and part of the linker region (shown in black) between the D/D to become disordered. This disordered region is required for PKA to undergo LLPS through which cAMP-dependent signaling is regulated. The figure was made based on the reported model in [6]. This image was created with Biorender.com

Figure 3.

Multivalent protein-protein interactions and PTMs drive LLPS of RTK and downstream substrates at the plasma membrane. Upon ligand binding, EGFR gets phosphorylated at multiple Tyr residues (pY) within its disordered C-terminal tail, creating binding sites to the adaptor protein Grb2 to recruit other signaling proteins that activate the MAPK pathway. Grb2 binds to pY residues at the C-terminal tail of EGFR via its SH2 domain. Then, the guanine nucleotide exchange factor SOS is recruited via interactions between its proline-rich (PR) domain and the SH3 domain of Grb2. These multiple interactions as well as PTMs in the EGFR promote the formation of phase separated condensates at the plasma membrane. Additional proteins are recruited to promote the activation of the MAPK signaling pathway. Other RTKs undergo LLPS in a similar manner, where PTMs initiate LLPS through modular protein interactions.

Figure 4.. Analysis of LLPS-prone sequences in oncogenic kinase fusions.

The sequences of each kinase and fusion partner were obtained from UniProt and combined for analysis by the online tool ParSe (http://folding.chemistry.msstate.edu/utils/parse.html). The breakpoint (▼) was obtained from Uniprot or estimated based on previous reports with other fusion partners. The mean ^rmodel in the human proteome and in the folded set used is shown with a dashed or a solid black line, respectively. ParSe-generated graphs were remade using GraphPad Prism 9 and the CSV data available for Protein Regions on ParSe. Black lines represent protein regions that may or may not be intrinsically disordered but that adopt a folded structure (F); red lines represent predicted intrinsically disordered regions that are not prone to undergo LLPS (D); and blue lines represent predicted intrinsically disordered regions that are prone to undergo LLPS (P). Regions that are not classified in one of these categories (G) are represented in gray. A) BCR-Abl includes the residues 1-426 from BCR and 27-1130 from Abl as reported on UniProt. B) EML4-ALK includes the residues 1-459 from EML4 and 1058-1620 from ALK as reported on Uniprot for variant 1. C) CCDC6-RET includes residues 1-101 from CCDC6 and 713-1114 from RET as reported on Uniprot. D) CEP85L-ROS1 includes residues 1-674 from CEP85L and 1881-2347 from ROS1 as reported on Uniprot as breakpoints for other fusions. E) KANK1-PDGFRB includes residues 1-1146 from KANK1 and 528-1106 from PDGFRβ as reported on Uniprot as breakpoints for other fusions.