Irene Cheng Jie Lee‡,§,1, Thomas Leung‡,§and Ivan Tan‡
‡ Institute of Molecular and Cell Biology, A-STAR, 61 Biopolis Drive, Singapore 138673
§ Department of Anatomy, National University of Singapore, Singapore 119260, Singapore
1 Present address: Signature Research Program in Cardiovascular and Metabolic
Disorders, Duke National University of Singapore Graduate Medical
School, Singapore 169857, Singapore.
Published in Journal of Biological Chemistry on 26 September 2014.
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 the activation of lamellar actomyosin network essential for cell migration. Here we report the identification of another adaptor protein LRAP25 that mediates MRCK association with LIMK1. The lamellipodium-localized LRAP25-MRCK complex is essential for the regulation of local LIM Kinase 1 (LIMK1) and its downstream F-actin regulatory factor cofilin. Functionally, inhibition of either MRCK or LRAP25 resulted in marked suppression of LIMK1 activity and downregulation 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 LRAP25-MRCK complex in LIMK1-cofilin signaling, and the importance of LRAP adaptors as key determinants of MRCK cellular localization and downstream specificities.
(A) Co-localization of GFP-MRCKa and b-actin-mCherry at the lamellipodia of B16-F1 cells treated with aluminum fluoride (AlF).
Scale bar, 10 mm.
(B) Depletion of LRAP25 resulted in down-regulation of FLAG-LIMK1 activity as reflected by the reductions in the level of
Thr-508 phosphorylation. Cells were treated with AlF as indicated. Lysates were immunoblotted with anti-FLAG, anti-pLIMK1(Thr(P)-508),
anti-LRAP25, or anti-tubulina antibodies.
(C) Depletion of LRAP25 resulted in reduced incorporation of fluorescently labelled actin
monomers into actin filaments at the leading edge, indicating a reduction in the density of free-barbed ends in the cell edge regions
resulted from perturbed local F-actin dynamics.
(D) Model of MRCK complex regulation of LIMK1 and cofilin. Rac activation in response to extracellular signal targets MRCK complex
to the cell membrane. Activation of MRCK by lipid hydrolysis (diacylglycerol) results in phosphorylation/activation of LIMK1, which in
turn causes phosphorylation/inactivation of the F-actin depolymerizing/severing factor cofilin. Activation of the cofilin phosphatase
SSH-1L in response to Rac activation results in dephosphorylation of cofilin. Coordination of the activities of the kinases and phosphatases
are required to maintain the dynamic phosphorylation regulation of cofilin that is important for lamellipodial F-actin regulation.
In the process of cell protrusion, MRCK translocates from the lamellipodium to the lamella with a corresponding change in complexity.
The translocation is believed to be important for coordinating cytoskeletal regulations taking place in the two connected regions.
Lp, lamellipodium; Lm, lamella; DAG, diacylglycerol.
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