Evaluating Calmodulin-Protein Interactions by Rapid Photoactivated Cross-Linking in Live Cells Metabolically Labeled with Photo-Methionine.
J Proteome Res
; 18(10): 3780-3791, 2019 10 04.
Article
en En
| MEDLINE
| ID: mdl-31483676
ABSTRACT
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 â¼60% of the high specificity group was affected by manipulations of Ca2+, compared with â¼20% 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.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Calmodulina
/
Proteínas
/
Células
/
Reactivos de Enlaces Cruzados
/
Metionina
Tipo de estudio:
Prognostic_studies
Límite:
Humans
Idioma:
En
Revista:
J Proteome Res
Asunto de la revista:
BIOQUIMICA
Año:
2019
Tipo del documento:
Article
País de afiliación:
Estados Unidos