Evaluating Calmodulin–Protein Interactions by Rapid Photoactivated Cross-Linking in Live Cells Metabolically Labeled with Photo-Methionine

This
work addresses the question of how the Ca2+ sensor
protein calmodulin shapes cellular responses to Ca2+ signals.
Proteins interacting with affinity tagged calmodulin were captured
by rapid (t1/2 ≈ 7 s) photoactivated
cross-linking under basal conditions, after brief removal of extracellular
Ca2+ and during a cytosolic [Ca2+] transient
in cells metabolically labeled with a photoreactive methionine analog.
Tagged adducts were stringently enriched, and captured proteins were
identified and quantified by LC–MS/MS. A set of 489 proteins
including 27 known calmodulin interactors was derived. A threshold
for fractional capture was applied to define a high specificity group
of 170 proteins, including 22 known interactors, and a low specificity
group of 319 proteins. Capture of ∼60% of the high specificity
group was affected by manipulations of Ca2+, compared with
∼20% of the low specificity group. This suggests that the former
is likely to contain novel interactors of physiological significance.
The capture of 29 proteins, nearly all high specificity, was decreased
by the removal of extracellular Ca2+, although this does
not affect cytosolic [Ca2+]. Capture of half of these was
unaffected by the cytosolic [Ca2+] transient, consistent
with high local [Ca2+]. These proteins are hypothesized
to reside in or near microdomains of high [Ca2+] supported
by the Ca2+ influx.