Protein kinase D intracellular localization and activity control kinase D-interacting substrate of 220-kDa traffic through a postsynaptic density-95/discs large/zonula occludens-1-binding motif.

Protein kinase D (PKD) controls protein traffic from the trans-Golgi network (TGN) to the plasma membrane of epithelial cells in an isoform-specific manner. However, whether the different PKD isoforms could be selectively regulating the traffic of their specific substrates remains unexplored. We identified the C terminus of the different PKDs that constitutes a postsynaptic density-95/discs large/zonula occludens-1 (PDZ)-binding motif in PKD1 and PKD2, but not in PKD3, to be responsible for the differential control of kinase D-interacting substrate of 220-kDa (Kidins220) surface localization, a neural membrane protein identified as the first substrate of PKD1. A kinase-inactive mutant of PKD3 is only able to alter the localization of Kidins220 at the plasma membrane when its C terminus has been substituted by the PDZ-binding motif of PKD1 or PKD2. This isoform-specific regulation of Kidins220 transport might not be due to differences among kinase activity or substrate selectivity of the PKD isoenzymes but more to the adaptors bound to their unique C terminus. Furthermore, by mutating the autophosphorylation site Ser(916), located at the critical position -2 of the PDZ-binding domain within PKD1, or by phorbol ester stimulation, we demonstrate that the phosphorylation of this residue is crucial for Kidins220-regulated transport. We also discovered that Ser(916) trans-phosphorylation takes place among PKD1 molecules. Finally, we demonstrate that PKD1 association to intracellular membranes is critical to control Kidins220 traffic. Our findings reveal the molecular mechanism by which PKD localization and activity control the traffic of Kidins220, most likely by modulating the recruitment of PDZ proteins in an isoform-specific and phosphorylation-dependent manner.

Regulation of microtubules by Rho GTPases in migrating cells.

Microtubules (MTs) contribute to cell polarization and migration, but the molecular mechanism involved are unknown. We have explored signalling pathways that generate specific changes in MTs arrays in wounded monolayers of fibroblasts. In earlier work, we found that Rho GTPase and its effector mDia, stimulate selective MT stabilization in the lamella, whereas Cdc42 and the MT motor protein dynein regulate MT organizing centre (MTOC) reorientation towards the leading edge. We have now found that the MT tip proteins EB1 and adenomatous polyposis coli protein (APC) function with mDia to stabilize MTs and interact directly with mDia. EB1, APC and mDia localize to the ends of stabilized MTs suggesting that they may contribute to capping of these MTs. Models of MTOC reorientation suggest that the MTOC moves in front of the nucleus by dynein pulling on MTs. In contrast, we find by directly imaging MTOC reorientation that the nucleus moves rearward while the MTOC remains stationary. Rearward nuclear movement is coupled to retrograde actin-myosin flow and is regulated by Cdc42 and its effector myotonic dystrophy kinase-related Cdc42-binding kinase. Dynein is not involved in nuclear movement, but is essential to maintain the MTOC at the cell centroid. These results show that there are two Cdc42 pathways that regulate MTOC reorientation.

Mutations in the neurofilament light gene linked to Charcot-Marie-Tooth disease cause defects in transport.

Neurofilament light gene mutations have been linked to a subset of patients with Charcot-Marie-Tooth disease, the most common inherited motor and sensory neuropathy. We have previously shown that Charcot-Marie-Tooth-linked mutant neurofilament light assembles abnormally in non-neuronal cells. In this study, we have characterized the effects of expression of mutant neurofilament light proteins on axonal transport in a neuronal cell culture model. We demonstrated that the Charcot-Marie-Tooth-linked neurofilament light mutations: (i) affect the axonal transport of mutant neurofilaments; (ii) have a dominant-negative effect on the transport of wild-type neurofilaments; (iii) affect the transport of mitochondria and the anterograde axonal transport marker human amyloid precursor protein; (iv) result in alterations of retrograde axonal transport and (v) cause fragmentation of the Golgi apparatus. Increased neuritic degeneration was observed in neuronal cells overexpressing neurofilament light mutants. Our results suggest that these generalized axonal transport defects could be responsible for the neuropathy in Charcot-Marie-Tooth disease.

EB1 and APC bind to mDia to stabilize microtubules downstream of Rho and promote cell migration.

Lysophosphatidic acid (LPA) stimulates Rho GTPase and its effector, the formin mDia, to capture and stabilize microtubules in fibroblasts. We investigated whether mammalian EB1 and adenomatous polyposis coli (APC) function downstream of Rho-mDia in microtubule stabilization. A carboxy-terminal APC-binding fragment of EB1 (EB1-C) functioned as a dominant-negative inhibitor of microtubule stabilization induced by LPA or active mDia. Knockdown of EB1 with small interfering RNAs also prevented microtubule stabilization. Expression of either full-length EB1 or APC, but not an APC-binding mutant of EB1, was sufficient to stabilize microtubules. Binding and localization studies showed that EB1, APC and mDia may form a complex at stable microtubule ends. Furthermore, EB1-C, but not an APC-binding mutant, inhibited fibroblast migration in an in vitro wounding assay. These results show an evolutionarily conserved pathway for microtubule capture, and suggest that mDia functions as a scaffold protein for EB1 and APC to stabilize microtubules and promote cell migration.

Lipid raft disruption triggers protein kinase C and Src-dependent protein kinase D activation and Kidins220 phosphorylation in neuronal cells.

Kidins220 (kinase D-interacting substrate of 220 kDa) is a novel neurospecific protein recently cloned as the first substrate for the Ser/Thr kinase protein kinase D (PKD). Herein we report that Kidins220 is constitutively associated to lipid rafts in PC12 cells, rat primary cortical neurons, and brain synaptosomes. Immunocytochemistry and confocal microscopy together with sucrose gradient fractionation show co-localization of Kidins220 and lipid raft-associated proteins. In addition, cholesterol depletion of cell membranes with methyl-beta-cyclodextrin dramatically alters Kidins220 localization and detergent solubility. By studying the putative involvement of lipid rafts in PKD activation and signaling we have found that active PKD partitions in lipid raft fractions after sucrose gradient centrifugation and that green fluorescent protein-PKD translocates to lipid raft microdomains at the plasma membrane after phorbol ester treatment. Strikingly, lipid rafts disruption by methyl-beta-cyclodextrin delays green fluorescent protein-PKD translocation, as determined by live cell confocal microscopy, and activates PKD, increasing Kidins220 phosphorylation on Ser(919) by a mechanism involving PKCepsilon and the small soluble tyrosine kinase Src. Collectively, these results reveal the importance of lipid rafts on PKD activation, translocation, and downstream signaling to its substrate Kidins220.

The neuronal protein Kidins220 localizes in a raft compartment at the leading edge of motile immature dendritic cells.

Kidins220, a protein predominantly expressed in neural tissues, is the first physiological substrate for protein kinase D (PKD). We show that Kidins220 is expressed in monocyte-derived and in peripheral blood immature dendritic cells (im DC). Immature DC (im DC) migrate onto extracellular matrices changing cyclically from a highly polarized morphology (monopolar (MP) stage) to a morphologically symmetrical shape (bipolar (BP) stage). Kidins220 was localized on membrane protrusions at the leading edge or on both poles in MP and BP cells, respectively. CD43, CD44, ICAM-3 and DC-SIGN, and signaling molecules PKD, Arp2/3 were found at the leading edge in MP or on both edges in BP cells, showing an intriguing parallelism between morphology and localization of molecular components on the poles of the motile DC. F-actin co-localized and it was necessary for Kidins220 localization on the membrane in MP and BP cells. Kidins220 was also found in a raft compartment. Disruption of rafts with methyl-beta-cyclodextrin induced rounding of the cells, inhibition of motility and lost of Kidins220 polarization. Our results describe for the first time the molecular components of the poles of motile im DC and indicate that a novel neuronal protein may be an important component among these molecules.