A Protein Kinase from Neutrophils That Specifically Recognizes Ser-3 in Cofilin

Jian P Lian,Peter G Marks,Jay Y Wang,Douglas L Falls,John A Badwey

doi:10.1074/jbc.275.4.2869

Jian P Lian, Peter G Marks + Show 3 more

Open Access

https://doi.org/10.1074/jbc.275.4.2869

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Abstract

Cofilin promotes the depolymerization of actin filaments, which is required for a variety of cellular responses such as the formation of lamellipodia and chemotaxis. Phosphorylation of cofilin on serine residue 3 is known to block these activities. We now report that neutrophils contain a protein kinase that selectively catalyzes the phosphorylation of cofilin on serine 3 (>/=70%) and a nonspecific kinase that recognizes multiple sites in this protein. The selective serine 3 cofilin kinase binds to a deoxyribonuclease I affinity column, whereas the nonspecific cofilin kinase does not. Deoxyribonuclease I forms a very tight complex with actin, and deoxyribonuclease affinity columns have been utilized to identify a variety of proteins that interact with the cytoskeleton. The serine 3 cofilin kinase did not react with antibodies to LIM kinase 1 or 2, which can catalyze the phosphorylation of cofilin in other cell types. The activity of the serine 3 cofilin kinase was insensitive to a variety of selective antagonists of protein kinases but was blocked by staurosporine. This pattern of inhibition is similar to that observed for the kinase that is active with cofilin in intact neutrophils. Thus, neutrophils contain a protein kinase distinct from LIM kinase-1/2 that selectively recognizes serine 3 in cofilin.

Highlights

Continuous polymerization and depolymerization of actin filaments is required for various cell responses such as the formation of lamellipodia, filopodia, and chemotaxis (e.g. Refs. 1–3)
Protein Kinases in Neutrophils That Catalyze Phosphorylation of Cofilin—Recombinant cofilin and [␥-32P]ATP served as substrates to search for protein kinases in neutrophils that were capable of catalyzing the phosphorylation of cofilin
The Ser-3 cofilin kinase (S3ck) we describe appears to be distinct from LIM kinase 1 (LIMK1) which recognizes cofilin in other cell types (24 – 26)

Summary

Introduction

Continuous polymerization and depolymerization of actin filaments is required for various cell responses such as the formation of lamellipodia, filopodia, and chemotaxis (e.g. Refs. 1–3). The NADPH-oxidase system catalyzes the production of large quantities of O2Ϫ/H2O2, which are key components of the oxygen-dependent, antimicrobial arsenal of these cells (e.g. Ref. 21) Consistent with these biochemical studies, neutrophils stimulated under certain conditions exhibit oscillations in O2Ϫ/H2O2 production that correlate with periodic extension and retraction of the lamellipodia and polymerization/depolymerization of actin [22, 23]. Recent reports have suggested that activated (GTP-bound) Rac mediates actin reorganization, in part, by stimulating LIM kinase 1 (LIMK1) [24, 25], which in turn catalyzes the phosphorylation/inactivation of cofilin (24 – 26). This pattern is not observed in neutrophils. This enzyme can be separated from a kinase(s) in these cells that recognizes multiple sites in cofilin (by DNase-agarose affinity chromatography) and appears to be distinct from fonamide; ML-7, 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4diazepine hydrochloride; KN-62, 1-[N,O-bis(1,5-isoquinolinesulfonyl)-Nmethyl-L-tyrosyl]-4-phenylpiperazine; GST, glutathione S-transferase; MBP, myelin basic protein; PAGE, polyacrylamide gel electrophoresis; Tricine, N-tris(hydroxymethyl)methylglycine

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