The role of Leucine Repeat Adaptor Protein 25(LRAP25), a novel interactor of Myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK) in mediating lamellipodial F-actin dynamics

Abstract

Migrating cells advance with sheet-like protrusive structures, which are essentially made up of distinct filamentous-actin (F-actin) networks, namely lamellipodia and lamella. Such protrusive advancement requires coordinated actin cytoskeletal rearrangements downstream of Rho family GTPases like Cdc42, Rho and Rac via the recruitment of various downstream effectors proteins. Myotonic dystrophy kinase-related Cdc42-binding kinases (MRCKs) are the multi-domained Serine/Threonine kinases downstream of Cdc42 and Rac1. MRCK plays vital roles in myosin dependent-contractility for the generation of traction force underlying cell motility. As a Cdc42 and Rac1 effector, MRCK thus responds to GTPase activation and recruited to the leading edge as suggested from previous reports. However, its role in the lamellipodia remains elusive. Therefore, having characterized MRCK as a key regulator in lamella retrograde actin flow via its adaptor protein, Leucine Repeat Adaptor Protein 35a (LRAP35a), it is important to understand its function and its underlying targeting mechanism to the lamellipodia. The current study provides evidence that MRCK together with its novel interactor, LRAP25 play an important role in regulating lamellipodial actin dynamics. MRCK function in the lamellipodia is established in the current study through its in vivo activation on LIM kinase 1 (LIMK1) activity and the subsequent regulation on the lamellipodial LIMK1 substrate, Cofilin. Inhibition of MRCK in B16F1 cells led to significant impairments in LIMK1 activation by AlF (an inducer of lamellipodia) and subsequent phosphorylation of Cofilin on Ser-3. Accordingly, LRAP25 depletion in B16F1 cells impaired MRCK recruitment to the lamellipodia and decreased lamellipodial pool of phosphorylated Cofilin on Ser-3. The collective findings of the mistargeting of MRCK from the lamellipodia and the perturbed leading edge actin dynamics observed upon LRAP25-depletion are highly suggestive of the deregulation of Cofilin activity. Taken together, the current study strongly suggests a crucial role of LRAP25 in localizing MRCK to ensure tight regulation on LIMK1, Cofilin and lamellipodial F-actin dynamics.