The 14-3-3ζ Protein Binds to the Cell Adhesion Molecule L1, Promotes L1 Phosphorylation by CKII and Influences L1-Dependent Neurite Outgrowth

Promotes L1 Phosphorylation by CKII and Influences L1-Dependent Neurite Outgrowth. PLoS ONE 5(10): e13462. doi:10.1371/journal.pone.0013462 The 14-3-3f Protein Binds to the Cell Adhesion Molecule L1, Promotes L1 Phosphorylation by CKII and Influences L1-Dependent Neurite Outgrowth Elisa M. Ramser 0 Gerrit Wolters 0 Galina Dityateva 0 Alexander Dityatev 0 Melitta Schachner 0 Thomas Tilling 0 Karl-Wilhelm Koch, University of Oldenburg, Germany 0 1 Zentrum fu r Molekulare Neurobiologie Hamburg, University of Hamburg , Hamburg, Germany , 2 Department of Neuroscience and Brain Technologies, Italian Institute of Technology , Genova , Italy , 3 Keck Center for Collaborative Neuroscience and Department of Cell Biology and Neuroscience, Rutgers University , Piscataway, New Jersey , United States of America Background: The cell adhesion molecule L1 is crucial for mammalian nervous system development. L1 acts as a mediator of signaling events through its intracellular domain, which comprises a putative binding site for 14-3-3 proteins. These regulators of diverse cellular processes are abundant in the brain and preferentially expressed by neurons. In this study, we investigated whether L1 interacts with 14-3-3 proteins, how this interaction is mediated, and whether 14-3-3 proteins influence the function of L1. Methodology/Principal Findings: By immunoprecipitation, we demonstrated that 14-3-3 proteins are associated with L1 in mouse brain. The site of 14-3-3 interaction in the L1 intracellular domain (L1ICD), which was identified by site-directed mutagenesis and direct binding assays, is phosphorylated by casein kinase II (CKII), and CKII phosphorylation of the L1ICD enhances binding of the 14-3-3 zeta isoform (14-3-3f). Interestingly, in an in vitro phosphorylation assay, 14-3-3f promoted CKII-dependent phosphorylation of the L1ICD. Given that L1 phosphorylation by CKII has been implicated in L1-triggered axonal elongation, we investigated the influence of 14-3-3f on L1-dependent neurite outgrowth. We found that expression of a mutated form of 14-3-3f, which impairs interactions of 14-3-3f with its binding partners, stimulated neurite elongation from cultured rat hippocampal neurons, supporting a functional connection between L1 and 14-3-3f. Conclusions/Significance: Our results suggest that 14-3-3f, a novel direct binding partner of the L1ICD, promotes L1 phosphorylation by CKII in the central nervous system, and regulates neurite outgrowth, an important biological process triggered by L1. - Funding: The study was partially supported by the German Federal Ministry of Education and Research, project no. 0311762/8 (to M.S.), and the New Jersey Commission for Spinal Cord Research (to M.S.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: M.S. is a consultant at the Center of Neuroscience of Shantou University Medical College, China. This does not alter the authors adherence to all the PLoS ONE policies on sharing data and materials. L1 is a cell adhesion molecule of the immunoglobulin superfamily which is essential for normal development of the mammalian nervous system. Constitutively L1-deficient mice display severe brain malformations, in particular hydrocephalus and agenesis of the corpus callosum [1,2]. Similar deficits have been discovered in humans carrying mutations in their L1CAM gene [3]. It has been demonstrated that cell recognition via L1 is important both for axon outgrowth and for neuronal migration (reviewed in [4,5]). These processes are likely to require dynamic control of L1-mediated cell adhesion, for instance by internalization of L1, regulating the availability of L1 on the cell surface. In support of this assumption, endocytotic trafficking of L1 has proved to be important for axon elongation [6]. Regulated L1 internalization depends on interactions of its intracellular domain with signaling, cytoskeletal, and adaptor molecules [7]. In particular, the tyrosine-based sorting motif Y1176RSL, which interacts with the adaptor protein AP-2, is necessary for clathrinmediated endocytosis of L1 [8]. Phosphorylation of Y1176 by the nonreceptor tyrosine kinase p60src prevents L1 binding to AP-2 [9]. This motif overlaps with the RSLE sequence, encoded by the alternatively spliced exon 28 [10]. The RSLE sequence is present only in L1 from neurons, but not in L1 expressed by non-neuronal cells such as Schwann cells [11]. Ser1181, the second serine residue of the YRSLESDNEE sequence in the L1ICD, can be phosphorylated by CKII [12]. This posttranslational modification most probably plays a critical role in endocytotic trafficking and L1-stimulated axon elongation [13]. However, molecular mechanisms by which CKIImediated phosphorylation could influence L1 function have not been investigated so far. Notably, the resulting RSLEpS sequence is a potential binding motif for 14-3-3 proteins [14], and analysis of transgenic mice ectopically expressing L1 in astrocytes (GFAP/L1 mice) [15] revealed an overexpression of 14-3-3b and f (T. Tilling et al., unpublished data). The 14-3-3 family of protein-binding proteins was first discovered in brain, where it comprises ,1% of total soluble protein [16]. 14-3-3 proteins are preferentially localized in neurons, but also expressed in a wide range of other cells and tissues [17]. The broad spectrum of 14-3-3 functions includes activation of tyrosine and tryptophan hydroxylases [18], regulation of the Raf-1 oncogene [1921], and modulation of apoptosis [22,23]. Consistent with their abundance in the brain, several studies point to an important role of 14-3-3 proteins in the nervous system. Genetic knock-out of 14-3-3 in Drosophila revealed an impairment of learning and synaptic plasticity [24]. In support of a similar function in mammals, Simsek-Duran et al. (2004) [25] have shown that 14-3-3 proteins are required for a presynaptic form of long-term potentiation in the mouse cerebellum. Moreover, members of the 14-3-3 family are involved in neuronal migration during vertebrate development [26], regulation of cerebellar NMDA receptor surface localization [27], and in neurotrophinstimulated growth of neurites [28,29]. The multitude of functions exerted by 14-3-3 proteins is achieved through their ability to bind to phosphoserine/phosphothreoninecontaining motifs of their ligands in a sequence specific manner. Two of the best known 14-3-3 consensus binding motifs are RSXpSXP and RXXXpSXP (pS represents the phosphorylated serine residue) [30]. However, 14-3-3 proteins not only recognize these classical motifs, but also other phosphorylated sites and nonphosphorylated motifs [14,31]. Owing to the versatility of binding sites in other proteins and to their ability to dimerize, 143-3 proteins act as adaptor proteins, chaperones and scaffolds [22]. For a better understanding of L1-mediated cell-cell interactions, it is crucial to elucidate th (...truncated)