Protein-Sequence-Based Search of Nonreceptor ITAM-Like Regions to Identify Cytosolic Syk-Recruiting Proteins

The
recruitment of the protein spleen tyrosine kinase (Syk) to
membrane-bound immune receptors is an essential step in initiating
an immune response mediated through the activated receptors. Syk recognizes
intracellular phosphorylated regions of membrane receptors known as
immunoreceptor tyrosine-based activation motifs (ITAMs) defined by
a sequence with two tyrosine (Y) amino acids separated by a certain
spacing of six to eight residues: YXX(I/L)X6–8YXX(I/L).
Syk with doubly phosphorylated ITAM is high-affinity and negatively
regulated when Syk itself becomes phosphorylated. While the role of
Syk in immune signaling is well characterized, recent information
affords new functionality to Syk related to cytoplasmic processes,
including the clearance of stress granules and P-bodies, both formed
by liquid–liquid phase separation. Little to nothing is known
about the molecular interactions involving Syk in these cytoplasmic
processes. Given the essential role of receptor ITAMs in recruiting
and localizing Syk for immune signaling, we explore here the possibility
of a similar localization mechanism occurring for cytoplasmic processes
by searching sequences of proteins related to Syk cytoplasmic function
for regions that resemble receptor ITAMs. Protein sequence databases
were generated from a Syk-dependent phosphoproteome and from genes
related to P-bodies. A search of these databases for ITAM-like sequences
yielded 102 unique hits, and 33 of these were synthesized and tested
experimentally for binding to Syk tandem SH2 domains. The equilibrium
dissociation constants were 0.1–50 μM for 28 peptides,
and binding was negatively regulated by phosphorylation for many peptides.
These results identify cytoplasmic proteins with potential for regulating
the localization of Syk in a phosphorylation-dependent manner to nonmembrane
cellular regions.