c-Jun N-Terminal Kinase Phosphorylation of MARCKSL1 Determines Actin Stability and Migration in Neurons and in Cancer Cells

Abstract

f Cell migration is a fundamental biological function, critical during development and regeneration, whereas deregulated migration underlies neurological birth defects and cancer metastasis. MARCKS-like protein 1 (MARCKSL1) is widely expressed in nervous tissue, where, like Jun N-terminal protein kinase (JNK), it is required for neural tube formation, though the mechanism is unknown. Here we show that MARCKSL1 is directly phosphorylated by JNK on C-terminal residues (S120, T148, and T183). This phosphorylation enables MARCKSL1 to bundle and stabilize F-actin, increase filopodium numbers and dynamics, and retard migration in neurons. Conversely, when MARCKSL1 phosphorylation is inhibited, actin mobility increases and filopodium formation is compromised whereas lamellipodium formation is enhanced, as is cell migration. We find that MARCKSL1 mRNA is upregulated in a broad range of cancer types and that MARCKSL1 protein is strongly induced in primary prostate carcinomas. Gene knockdown in prostate cancer cells or in neurons reveals a critical role for MARCKSL1 in migration that is dependent on the phosphorylation state; phosphomimetic MARCKSL1 (MARCKSL1 S120D,T148D,T183D ) inhibits whereas dephospho-MARCKSL1 S120A,T148A,T183A induces migration. In summary, these data show that JNK phosphorylation of MARCKSL1 regulates actin homeostasis, filopodium and lamellipodium formation, and neuronal migration under physiological conditions and that, when ectopically expressed in prostate cancer cells, MARCKSL1 again determines cell movement. M ARCKS-like protein 1 (MARCKSL1) is an actin binding protein that is predominantly expressed in immature brain (1, 27). The MARCKSL1 homologue MARCKS has been more extensively studied and has been shown to bind actin with a stoichiometry of 1:2, thereby facilitating cross-linking (56; reviewed in reference 39). Binding to actin occurs via an effector domain (ED) that is 87% identical to the corresponding domain of MARCKSL1. Surprisingly, however, full-length MARCKSL1 does not cross-link F-actin (49; reviewed in reference 39), although the MARCKSL1 effector domain alone interacts with actin. This indicates that in a physiological context, another level of regulation is required for MARCKSL1 to regulate actin bundling. The only known critical function of MARCKSL1 is in early development of the nervous system, as genetic disruption of MARCKSL1 results in neural tube closure defects (5, 51), events that depend on coordinated control of actin functions, cell shape, and cell migration. MARCKSL1 is also associated with cell spreading (23); however, the mechanism whereby MARCKSL1 regulates F-actin in a cellular context has remained obscure.c-Jun N-terminal kinase 1 (JNK1) and JNK2, like MARCKSL1, are required for neural tube closure (20,36). However, the identity of the JNK effectors mediating this event remains unresolved. JNK activity is highly elevated in neuronal cells (8,42), and although initially unexpected (7,8,53), it is now accepted that a number of important physiological substrates fo...

JNK Signaling: Regulation and Functions Based on Complex Protein-Protein Partnerships

“…Lastly, the fully disordered actin-binding protein MARCSKL1 is another important cytoskeletal JNK substrate. In this case, concomitant phosphorylation of three sites (corresponding to Ser120, Thr148, and Thr178 in human MARCKSL1) enhanced the F-actin-binding capacity (266). Two of the three sites are poorly conserved, but Ser120 lies in a region (SSPTEEEQ) also evident in its fish orthologs.…”

“…The compound disruption of both the MKK4 and MKK7 genes results in severe growth retardation and embryonic lethality (E9.5). It is striking that genetic deletion of single JNK substrates may also phenocopy JNK1 Ϫ/Ϫ JNK2 Ϫ/Ϫ mice, as observed for mice lacking the actin regulatory protein MARCKS-like protein 1 (266). While MARCKS-like protein 1 gene deletion causes neural tube defects (391), knockout mice for c-Jun or ATF2 live longer but die of heart failure or respiratory failure, respectively (392,393).…”

“…S1C). These cytoskeletal JNK substrate proteins include those associating with microtubules (DCX [260][261][262], MAP1B [180,263], MAP2 [264], Tau [265], and WDR62 [213]), the actin cytoskeleton (MARCKSL1 [266] and SMTL2 [267]), or focal adhesions (paxillin [268,269] and ␤-catenin [270][271][272]). In addition, JNK-mediated phosphorylation of additional proteins may also direct vesicular transport (through phosphorylation of JIP1 and ␤-APP [104,217]), as well as the exocytosis of specialized, Glut4-containing vesicles (through insulin-stimulated phosphorylation of IRS1 and IRS2 [273,274]).…”

MARCKS regulates membrane targeting of Rab10 vesicles to promote axon development

“…Genetic studies show that MARCKS plays an important role in the development of the central nervous system, including neurulation, fusion of the cerebral hemispheres, formation of the forebrain commissures, and lamination of the cortex and retina [14]. Moreover, MARCKS is critical for neurite initiation, dendritic branching, spine maintenance [15][16][17], growth cone adhesion and path finding [18], and neuronal migration [19,20]. However, the direct linkage between the biochemical properties of MARCKS and its cellular functions has not been well established.…”

Quantitative Proteomics Reveals Dynamic Interaction of c-Jun N-terminal Kinase (JNK) with RNA Transport Granule Proteins Splicing Factor Proline- and Glutamine-rich (Sfpq) and Non-POU Domain-containing Octamer-binding Protein (Nono) during Neuronal Differentiation

“…Last, P19-differentiated neurons were enzymatically dissociated and plated in DMEM containing 5% FBS and 100 g/ml penicillin/streptomycin. Neurons were cultured for 4,6,8,12,24,36, and 48 h.…”