C2 domains are widely-spread protein signaling motifs that in classical PKCs act as Ca ²⁺ -binding modules. However, the molecular mechanisms of their targeting process at the plasma membrane remain poorly understood. Here, the crystal structure of PKCα-C2 domain in complex with Ca ²⁺ , 1,2-dihexanoyl- sn -glycero-3-[phospho- l -serine] (PtdSer), and 1,2-diayl- sn -glycero-3-[phosphoinositol-4,5-bisphosphate] [PtdIns( 4 , 5 )P ₂ ] shows that PtdSer binds specifically to the calcium-binding region, whereas PtdIns( 4 , 5 )P ₂ occupies the concave surface of strands β3 and β4. Strikingly, the structure reveals a PtdIns( 4 , 5 )P ₂ -C2 domain-binding mode in which the aromatic residues Tyr-195 and Trp-245 establish direct interactions with the phosphate moieties of the inositol ring. Mutations that abrogate Tyr-195 and Trp-245 recognition of PtdIns( 4 , 5 )P ₂ severely impaired the ability of PKCα to localize to the plasma membrane. Notably, these residues are highly conserved among C2 domains of topology I, and a general mechanism of C2 domain-membrane docking mediated by PtdIns( 4 , 5 )P ₂ is presented.

Charcot–Marie–Tooth (CMT) neuropathies are highly heterogeneous disorders caused by mutations in more than 70 genes, with no available treatment. Thus, it is difficult to envisage a single suitable treatment for all pathogenetic mechanisms. Axonal Neuregulin 1 (Nrg1) type III drives Schwann cell myelination and determines myelin thickness by ErbB2/B3‐PI3K–Akt signaling pathway activation. Nrg1 type III is inhibited by the α‐secretase Tace, which negatively regulates PNS myelination. We hypothesized that modulation of Nrg1 levels and/or secretase activity may constitute a unifying treatment strategy for CMT neuropathies with focal hypermyelination as it could restore normal levels of myelination. Here we show that in vivo delivery of Niaspan, a FDA‐approved drug known to enhance TACE activity, efficiently rescues myelination in the Mtmr2 −/− mouse, a model of CMT4B1 with myelin outfoldings, and in the Pmp22 +/− mouse, which reproduces HNPP (hereditary neuropathy with liability to pressure palsies) with tomacula. Importantly, we also found that Niaspan reduces hypermyelination of Vim (vimentin)−/− mice, characterized by increased Nrg1 type III and Akt activation, thus corroborating the hypothesis that Niaspan treatment downregulates Nrg1 type III signaling. ![][1] The α‐secretase TACE negatively regulates Neuregulin 1 (Nrg1) type III, a main driver of Schwann cell myelination. Enhancement of TACE activity with Niaspan/niacin reduces focal hypermyelination in Charcot–Marie–Tooth and HNPP neuropathy mouse models. [1]: /embed/graphic-1.gif

Significance: Vesicle fusion is an important event in neuronal transmission and endocrine cell secretion. A myriad of proteins containing double C2 domains are involved in this complex process; however, how Ca ²⁺ and the different types of membrane lipids regulate their function is still not well understood. In this work, we provide structural insights to explain the ability of different C2 domains to interact with Ca ²⁺ and PI(4,5)P ₂ and demonstrate the existence of a specific PI(4,5)P ₂ -binding motif that provides these domains with specific properties to interact with the membrane and initiate vesicle fusion. We also demonstrate a unique molecular mechanism conferring their specificity for the different phosphoinositides, which resides in additional amino acidic residues surrounding the key interacting lysines.

Microtubule-based kinesin motors have many cellular functions, including the transport of a variety of cargos. However, unconventional roles have recently emerged, and kinesins have also been reported to act as scaffolding proteins and signaling molecules. In this work, we further extend the notion of unconventional functions for kinesin motor proteins, and we propose that Kif13b kinesin acts as a signaling molecule regulating peripheral nervous system (PNS) and central nervous system (CNS) myelination. In this process, positive and negative signals must be tightly coordinated in time and space to orchestrate myelin biogenesis. Here, we report that in Schwann cells Kif13b positively regulates myelination by promoting p38γ mitogen-activated protein kinase (MAPK)-mediated phosphorylation and ubiquitination of Discs large 1 (Dlg1), a known brake on myelination, which downregulates the phosphatidylinositol 3-kinase (PI3K)/v-AKT murine thymoma viral oncogene homolog (AKT) pathway. Interestingly, Kif13b also negatively regulates Dlg1 stability in oligodendrocytes, in which Dlg1, in contrast to Schwann cells, enhances AKT activation and promotes myelination. Thus, our data indicate that Kif13b is a negative regulator of CNS myelination. In summary, we propose a novel function for the Kif13b kinesin in glial cells as a key component of the PI3K/AKT signaling pathway, which controls myelination in both PNS and CNS. Myelin is a multilayered extension of the Schwann and oligodendrocyte cell membranes, which wraps around neuronal axons to facilitate propagation of electric signals and to support axonal metabolism. However, the signals regulating myelin formation and how they are integrated and controlled to achieve homeostasis are still poorly understood. In Schwann cells, the Discs large 1 (Dlg1) protein is a known brake of myelination, which negatively regulates the amount of myelin produced so that myelin thickness is proportional to axonal diameter. In this paper, we report that in Schwann cells Dlg1 itself is tightly regulated to ensure proper myelination. We propose that Dlg1 function is further controlled by the Kif13b kinesin motor protein, which acts as a "brake of the brake" by downregulating Dlg1 activity. Surprisingly, we found that in oligodendrocytes Dlg1 is a positive and not a negative regulator of myelination. Thus, Kif13b-mediated negative regulation of Dlg1 ensures appropriate myelin production and thickness in the central nervous system. Our data further extend recently emerged unconventional roles for kinesins, which are usually implicated in cargo transport rather than in the modulation of signaling pathways. The elucidation of mechanisms regulating myelination may help to design specific approaches to favor re-myelination in demyelinating disorders in which this process is severely impaired.

Significance: Charcot-Marie-Tooth type 4B1 (CMT4B1) is a very severe demyelinating neuropathy with childhood onset, characterized by myelin outfoldings, a pathological aberrant form of myelination. This morphology may be the consequence of an excessive longitudinal growth of the myelinated internode during postnatal nerve development. We first demonstrated that loss of MTMR2 (Myotubularin-related 2) phosphatase causes CMT4B1. MTMR2 dephosphorylates phospholipids, important regulators of membrane trafficking, which is a key process in Schwann cells, the myelinating cells in the PNS. However, why loss of MTMR2 provokes CMT4B1 remains to be assessed. Here we propose a mechanism by which MTMR2, by regulating PtdIns(3,5)P₂phosphoinositide levels, coordinates myelin synthesis and RhoA/myosin II-dependent cytoskeletal dynamics necessary to expand the membrane and promote myelin growth.