Protein phosphatase 1delta (PP1delta) localizes to focal adhesions and associates with the focal adhesion kinase (FAK). In the present work we used deletion mutants of PP1delta and FAK to detect their reciprocally interacting domains. Dissection of PP1delta indicated 194–260 as the shortest FAK-interacting domain among those tested. Domain 194–260 encompasses several sites involved in catalysis, indirectly confirming that FAK is a PP1 substrate. Mutation of one of these sites, R220 (R220S or R220Q), did not abolish but on the contrary increased the ability of 194–260 to pull-down FAK. Such property might be exploited to detect new potential PP1 substrates. Among the FAK deletion mutants, only the C-terminal domain (684–1053, also known as FRNK) pulled-down a significant amount of PP1. The PP1 eluted from a GST-FRNK affinity column displayed Mr of 35,000 when analyzed by gel-filtration on FPLC Superose 12, indicating the presence of an isolated PP1 catalytic subunit.

Reciprocally interacting domains of protein phosphatase 1 and focal adhesion kinase

DE LUCCHINI, STEFANIA;
2005

Abstract

Protein phosphatase 1delta (PP1delta) localizes to focal adhesions and associates with the focal adhesion kinase (FAK). In the present work we used deletion mutants of PP1delta and FAK to detect their reciprocally interacting domains. Dissection of PP1delta indicated 194–260 as the shortest FAK-interacting domain among those tested. Domain 194–260 encompasses several sites involved in catalysis, indirectly confirming that FAK is a PP1 substrate. Mutation of one of these sites, R220 (R220S or R220Q), did not abolish but on the contrary increased the ability of 194–260 to pull-down FAK. Such property might be exploited to detect new potential PP1 substrates. Among the FAK deletion mutants, only the C-terminal domain (684–1053, also known as FRNK) pulled-down a significant amount of PP1. The PP1 eluted from a GST-FRNK affinity column displayed Mr of 35,000 when analyzed by gel-filtration on FPLC Superose 12, indicating the presence of an isolated PP1 catalytic subunit.
2005
focal adhesion kinase - focal adhesions - phosphatase 1 - protein kinase - protein phosphatase
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11384/851
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