Entry created on 1 July 2019 (Revision 1.0) Annotator: Rita Pancsa
This entry is part of a multi-component system encompassing the following entries: O60500 O00401 P16333
Basic protein information
Accession P16333
Common name NCK1, Nck
Gene NCK1
Organism Homo sapiens
Uniprot name Cytoplasmic protein NCK1
Basic LLPS information
Organelle membrane cluster; actin cortical patch; Arp2/3 protein complex
Type of experimental evidence
Joined entry O60500 O00401 P16333
Protein region(s) mediating LLPS
1
-
377
Full protein sequence contributes to LLPS: SH3-SH3-SH3-SH2 domains with 50 residue conserved linker between SH3_1 and SH3_2
Based on the experimental results of the following publication: 26553976
Molecular features viewer
PDB structures
Extended LLPS information
Functional description
In kidney podocytes, the transmembrane protein nephrin plays a central role in forming the glomerular filtration barrier, functioning partly through assembling cortical actin. The cytoplasmic tail of nephrin contains three tyrosine phosphorylation (pTyr) sites, which can each bind the SH2 domain of Nck1. Nck contains three SH3 domains, which can bind the six PRMs in the proline-rich region of N-WASP. N-WASP, in turn, stimulates the nucleation of actin filaments by the Arp2/3 complex. The multivalency of nephrin or NCK is necessary for proper actin assembly and, together with the multivalency of N-WASP, has the potential to cause phase transitions (PMID:22398450, PMID:25321392). With nephrin attached to the bilayer, multivalent interactions enable these proteins to polymerize on the membrane surface and undergo two-dimensional phase separation, producing micrometer-sized clusters. Phosphorylated tyrosines of nephrin cytoplasmic domain get bound by the SH2 domain of Nck1 (PMID:22398450, PMID:25321392), but the NICD of nephrin is also able to form micron-scale nuclear bodies/liquid droplets on its own by complex coacervation helped by positively charged partners as well, even when the Ys are replaced by Fs, so no phosphorylation can happen (PMID:27392146). Also, the 50-residue linker between the first two SH3 domains of Nck enhances phase separation of Nck/N-WASP/nephrin assemblies (PMID:26553976). In the presence of the Arp2/3 complex, the clusters assemble actin filaments, suggesting that clustering of regulatory factors could promote local actin assembly at membranes (PMID:25321392). LLPS increases the specific activity of actin regulatory proteins toward actin assembly by the Arp2/3 complex. This increase occurs because LLPS of the Nephrin-Nck-N-WASP signaling pathway on lipid bilayers increases membrane dwell time of N-WASP and Arp2/3 complex, consequently increasing actin assembly. Dwell time varies with relative stoichiometry of the signaling proteins in the phase-separated clusters, rendering N-WASP and Arp2/3 activity stoichiometry dependent (PMID:30846599).
Literature supporting the LLPS: 22398450, 25321392, 26553976, 27392146, 30846599
Functional class of membraneless organelle: activation/nucleation/signal amplification/bioreactor; regulator of spatial patterns
Binding partners (at biological protein concentrations)
1) Nephrin (strictly required) 2) N-WASP (strictly required)
Type of RNA(s) required/used for the LLPS at biological protein concentrations
RNA not required.
Molecular interaction types contributing to LLPS
multivalent domain-motif interactions (PMID:22398450, PMID:25321392) multivalent domain-PTM interactions (PMID:22398450, PMID:25321392) complex coacervation (PMID:27392146)
Determinants of phase separation and droplet properties
1) phosphorylation state 2) valency of Nck1 3) valency of N-WASP 4) molecular affinities between the components 5) stoichiometry of the components
Membrane cluster Yes
Partner-dependent Yes
RNA-dependent No
PTM required Yes
Domain-motif interactions Yes
Discrete oligomerization No
Regulation and disease
Post-translational modifications affecting LLPS
Position Residue PTM Effect Reference Modifying enzyme Notes
Isoforms known to affect LLPS
Isoform Effect Reference
All known isoforms containing sequence changes in the LLPS region(s)
Position type Isoform names from UniProt
Disease mutations affecting LLPS
Mutation dbSNP Disease OMIM Effect Reference Notes
Experimental information
Experimental techniques applied to prove/investigate LLPS
In vitro experiments with engineered proteins: one composed of repeats of a single SH3 domain (SH3m, where m = 1–5), and the other composed of repeats of a PRM ligand (PRMn, where n = 1–5) showed change in optical properties (turbidity) due to the formation liquid droplets by phase separation as assessed by microscopy at high protein concentrations (change in protein concentration). The proteins were concentrated by about 100-fold in the droplets relative to the bulk phase. Higher valency (mutation) allowed for the formation of larger species (particle size and count) at a lower fractional saturation of the binding modules. The phase transition could be blocked by a high-affinity monovalent ligand. The multivalent proteins formed large polymers within the droplets (DLS, SAXS), such that the phase transition probably coincides with a sol–gel transition. The photobleaching recovery rate (FRAP) correlated inversely with the monomer–monomer affinity and valency, suggesting that recovery represents reorganization of a polymer matrix. The coexpression of mCherry–SH35 and eGFP–PRM5 fusion proteins in HeLa cells resulted in the formation of approximately 0.5–2-µm diameter (particle size and count) cytoplasmic (protein localization) puncta containing both fluorophores (protein co-localization) in vivo. The puncta did not stain with a large range of vesicle markers or a lipid dye, suggesting that they are phase-separated bodies rather than vesicular structures (morphology). The addition of NCK to an N-WASP construct caused droplet formation, as occurred in the model systems described above. The addition of a diphosphorylated (2pTyr) nephrin tail peptide dropped the phase boundary for both proteins by more than or equal to twofold (protein phosphorylation). This effect was even more pronounced when nephrin–3pTyr peptide (protein phosphorylation) was added (to the same total pTyr concentration), showing the importance of valency of the components and that the whole system could be regulated by kinases and phosphatases in vivo (PMID:22398450). Fluorescently tagged p-Nephrin, Nck and N-WASP co-localized to clusters formed on fluid supported lipid bilayers. Addition of 10 µM of a monovalent pTyr peptide derived from TIR (with KD of 40 nM for the Nck SH2 domain) to clusters formed from p-Nephrin /(SH3)3/N-WASP dissolved the clusters (particle size and count). Fluorescently tagged p-Nephrin (2200 molecules/µm²) was clustered by addition of 2 μM N-WASP and 1 μM Nck, addition of 10 nM Arp2/3 complex and 1 µM actin (10% rhodamine labeled) showed that actin specifically assembles on p-Nephrin/Nck/N-WASP clusters in an Arp2/3 dependent manner (protein co-localization) (PMID:25321392).
Experimental observations supporting the liquid material state of the condensate
morphological traits (PMID:22398450, PMID:25321392) dynamic movement/reorganization of molecules within the droplet (PMID:22398450, PMID:25321392) dynamic exchange of molecules with surrounding solvent (PMID:22398450)