Abstract
Myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK) has been shown to localize to the lamella of mammalian cells through its interaction with an adaptor protein, leucine repeat adaptor protein 35a (LRAP35a), which links it with myosin 18A (MYO18A) for activation of the lamellar actomyosin network essential for cell migration. Here, we report the identification of another adaptor protein LRAP25 that mediates MRCK association with LIM kinase 1 (LIMK1). The lamellipodium-localized LRAP25-MRCK complex is essential for the regulation of local LIMK1 and its downstream F-actin regulatory factor cofilin. Functionally, inhibition of either MRCK or LRAP25 resulted in a marked suppression of LIMK1 activity and down-regulation of cofilin phosphorylation in response to aluminum fluoride induction in B16-F1 cells, which eventually resulted in deregulation of lamellipodial F-actin and reorganization of cytoskeletal structures causing defects in cell polarization and motility. These biochemical and functional characterizations thus underline the functional relevance of the LRAP25-MRCK complex in LIMK1-cofilin signaling and the importance of LRAP adaptors as key determinants of MRCK cellular localization and downstream specificities.
Highlights
LIM kinase 1 (LIMK1) regulates F-actin dynamics through phosphorylation/inactivation of cofilin
Identification of leucine repeat adaptor protein 25 (LRAP25) as a Novel Interactor of Myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK)— Our previous work detected two proteins of 35 and 25 kDa in the MRCK immunoprecipitate [25]. The former was characterized as an adaptor protein leucine repeat adaptor protein 35a (LRAP35a) that is involved in regulating the kinase-dependent nonmuscle myosin II activity [25]
MRCK and LRAP25 Regulate LIMK1 Activity and Lamellipodial Localization in Vivo—As MRCK is known to activate LIMK1 [22], we examined whether it is required for LIMK1 activity that is implicated in lamellipodial formation [15, 31]
Summary
LIMK1 regulates F-actin dynamics through phosphorylation/inactivation of cofilin. Results: LRAP25 forms a complex with MRCK and LIMK1 to promote LIMK1 phosphorylation/activation by MRCK. Inhibition of either MRCK or LRAP25 resulted in a marked suppression of LIMK1 activity and down-regulation of cofilin phosphorylation in response to aluminum fluoride induction in B16-F1 cells, which eventually resulted in deregulation of lamellipodial F-actin and reorganization of cytoskeletal structures causing defects in cell polarization and motility These biochemical and functional characterizations underline the functional relevance of the LRAP25-MRCK complex in LIMK1-cofilin signaling and the importance of LRAP adaptors as key determinants of MRCK cellular localization and downstream specificities. Activation of Arp2/3 complex is achieved by the binding of nucleation-promoting factors such as Wiskott-Aldrich syndrome protein (WASP) and WASP-family verprolinhomologous (WAVE) proteins that are themselves downstream effectors of the small GTPases Rac and Cdc42 [4, 8] This actin polymerization of the fast growing barbed end filaments at the tip of lamellipodia is kept in balance by the depolymerization of pointed end actin filaments located at the base of the network [4, 9]. This study delineates a signaling cascade that involves LRAP25-MRCK and LIMK1-cofilin in the regulation of lamellipodial F-actin dynamics important for cell protrusion and migration
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