Signaling proteins
often form biomolecular condensates through
liquid–liquid phase separation (LLPS) during intracellular
signal transduction. Modulating the LLPS property of intracellular
protein condensates will redirect intracellular signals and provide
a potential way to regulate cellular physiology. Phosphorylation of
multiple tyrosine residues of the transmembrane receptor nephrin is
known to drive the LLPS of the adaptor protein Nck and neuronal Wiskott–Aldrich
Syndrome protein (N-WASP) and form the Nck signaling complex. Phosphorylation
of the translocated intimin receptor (Tir) in the host cell may recruit
this enteropathogenic Escherichia coli (EPEC) virulence
factor to the Nck signaling complex and lead to the entry of EPEC
into the intestine cell. In this work, we first identified a phosphotyrosine
(pY)-containing peptide 3pY based on the sequence similarity
of nephrin and Tir; 3pY promoted the LLPS of Nck and
N-WASP, mimicking the role of phosphorylated nephrin. Next, we designed
a covalent blocker of Nck, peptide p1 based on the selected
pY peptides, which site-selectively reacted with the SH2 domain of
Nck (Nck-SH2) at Lys331 through a proximity-induced reaction. The
covalent reaction of p1 with Nck blocked the protein
binding site of Nck-SH2 and disintegrated the 3pY/Nck/N-WASP
condensates. In the presence of membrane-translocating peptide L17E, p1 entered Caco-2 cells in the cytosol, reduced the number
of Nck puncta, and rendered Caco-2 cells resistant to EPEC infection.
Site-selective covalent blockage of Nck thereby disintegrates intracellular
Nck condensates, inhibits actin reorganization, and shuts down the
entrance pathway of EPEC. This work showcases the promotion or inhibition
of protein phase separation by synthetic peptides and the use of reactive
peptides as LLPS disruptors and signal modulators.