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.

Protein kinase D (PKD) is a serine/threonine kinase regulated by diacylglycerol signaling pathways with unique domain composition and enzymatic properties, still awaiting identification of its specific substrate(s). Here we have isolated, cloned, and characterized a novel protein from PC12 cells, termed Kidins220 (kinase D-interacting substrate of 220 kDa), as the first identified PKD physiological substrate. Kidins220 contains 11 ankyrin repeats and four transmembrane domains within the N-terminal region. We have shown that Kidins220 is an integral membrane protein selectively expressed in brain and neuroendocrine cells, where it concentrates at the tip of neurites. In PC12 cells, PKD coimmunoprecipitates and phosphorylates endogenous Kidins220. This phosphorylation is increased after stimulating PKD activity in vivo by phorbol-12,13-dibutyrate treatment. A constitutively active PKD mutant (PKD-S744E/S748E) phosphorylates recombinant Kindins220-VSVG in vitro in the absence of phorbol-12,13-dibutyrate. Conversely, Kidins220-VSVG phosphorylation is abolished when a dominant negative mutant of PKD (PKD-D733A) is used. Moreover, a peptide within the Kidins220 sequence, containing serine 919 in a consensus motif for PKD-specific phosphorylation, behaved as the best peptide substrate to date. Substitution of serine 919 to alanine abrogated peptide phosphorylation. Furthermore, by generating an antibody recognizing Kidins220 phosphorylated on serine 919, we show that phorbol ester treatment causes the specific phosphorylation of this residue in PC12 cells in vivo. Our results provide the first physiological substrate for PKD and indicate that Kidins220 is phosphorylated by PKD at serine 919 in vivo.Stimulation of several plasma membrane receptors by hormones, growth factors, and cytokines causes the rapid hydrolysis of phosphoinositides, which results in an increase of the lipid-derived second messenger diacylglycerol (DAG) 1 (1). DAG causes the activation of protein kinase C (PKC) (2, 3), which in turn affects several cellular processes including cell growth and differentiation, changes in cell morphology, neuronal development, and endocrine and exocrine secretion (2, 4 -6). PKC isoforms are divided into three major groups on the basis of their primary structure and biochemical properties (7-9). The conventional PKCs (␣, ␤1, ␤2, and ␥) contain a regulatory cysteine-rich domain that binds DAG and phorbol esters and a calcium binding domain responsible for the calcium-dependent modulation of their kinase activity. The novel PKCs (␦, ⑀, , and ) are sensitive to DAG or phorbol esters but are calciumindependent. The third group is known as the atypical PKCs ( and /), which are unresponsive to calcium, DAG, or phorbol esters.Protein kinase D (PKD, also known as PKC) is a serine/ threonine kinase distantly related to the PKC family. It contains a conserved DAG/phorbol ester-binding cysteine-rich domain like the classical and novel PKC isoforms but has singular enzymological and structural properties (10 -13). Unlike all oth...

During mitosis, the ribbon of the Golgi apparatus is transformed into dispersed tubulo-vesicular membranes, proposed to facilitate stochastic inheritance of this low copy number organelle at cytokinesis. Here, we have analyzed the mitotic disassembly of the Golgi apparatus in living cells and provide evidence that inheritance is accomplished through an ordered partitioning mechanism. Using a Sar1p dominant inhibitor of cargo exit from the endoplasmic reticulum (ER), we found that the disassembly of the Golgi observed during mitosis or microtubule disruption did not appear to involve retrograde transport of Golgi residents to the ER and subsequent reorganization of Golgi membrane fragments at ER exit sites, as has been suggested. Instead, direct visualization of a green fluorescent protein (GFP)-tagged Golgi resident through mitosis showed that the Golgi ribbon slowly reorganized into 1–3-μm fragments during G2/early prophase. A second stage of fragmentation occurred coincident with nuclear envelope breakdown and was accompanied by the bulk of mitotic Golgi redistribution. By metaphase, mitotic Golgi dynamics appeared to cease. Surprisingly, the disassembly of mitotic Golgi fragments was not a random event, but involved the reorganization of mitotic Golgi by microtubules, suggesting that analogous to chromosomes, the Golgi apparatus uses the mitotic spindle to ensure more accurate partitioning during cytokinesis.

We have examined the fate of Golgi membranes during mitotic inheritance in animal cells using four-dimensional fluorescence microscopy, serial section reconstruction of electron micrographs, and peroxidase cytochemistry to track the fate of a Golgi enzyme fused to horseradish peroxidase. All three approaches show that partitioning of Golgi membranes is mediated by Golgi clusters that persist throughout mitosis, together with shed vesicles that are often found associated with spindle microtubules. We have been unable to find evidence that Golgi membranes fuse during the later phases of mitosis with the endoplasmic reticulum (ER) as a strategy for Golgi partitioning (Zaal, K.J., C.L. Smith, R.S. Polishchuk, N. Altan, N.B. Cole, J. Ellenberg, K. Hirschberg, J.F. Presley, T.H. Roberts, E. Siggia, et al. 1999. Cell. 99:589–601) and suggest that these results, in part, are the consequence of slow or abortive folding of GFP–Golgi chimeras in the ER. Furthermore, we show that accurate partitioning is accomplished early in mitosis, by a process of cytoplasmic redistribution of Golgi fragments and vesicles yielding a balance of Golgi membranes on either side of the metaphase plate before cell division.

Protein kinase D (PKD) controls protein traffic from the transGolgi 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 phosphorylationdependent manner. Protein kinase D1 (PKD1)5 (1), also known as PKC (2), belongs to a novel family of diacylglycerol (DAG)-stimulated Ser/Thr kinases, composed of two more members PKD2 (3) and PKD3 (4) (reviewed in Refs. 5-7). PKDs contain several well characterized domains, including two cysteine-rich repeats (C1a and C1b) that constitute a C1 domain (CR), a pleckstrin homology domain, and a catalytic domain at the C terminus. The CR domain binds DAG and phorbol esters with high affinity (1,8) and is involved in the association of PKD1 to cellular membranes such as the plasma membrane and the trans-Golgi network (TGN) (9 -12). The pleckstrin homology domain is an autoinhibitory domain that regulates the activity of this kinase (13). PKD is activated by the phosphorylation of two activation loop sites within the catalytic domain through a protein kinase C (PKC)-dependent pathway, which stabilizes the enzyme in an active conformation (14, 15). Activated PKD1 autophosphorylates at Ser 916 present at the very C terminus, and this phosphorylation event is frequently used to determine the activation state of this kinase (16 -18).PKD has been shown to participate in many cellular processes, such as cell survival, proliferation, and invasion (5-7), but the regulation of protein transpo...

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.

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-␤-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-␤-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 PKC⑀ 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. Kidins220 (kinase D-interacting substrate of 220 kDa) 1 (1), also known as ankyrin repeat-rich membrane spanning or ARMS (2), is a novel integral membrane protein mainly expressed in brain, encoded by a single gene in Drosophila melanogaster, Caenorhabditis elegans, and mammals. Its primary amino acid sequence contains several structural and functional domains and diverse motifs that may link this protein to membranes, cytoskeleton, and different signaling pathways. The N terminus bears 11 ankyrin repeats that are likely to be involved in protein-protein interactions specially with the cytoskeleton (3). Downstream, the sequence presents four transmembrane domains and a proline-rich region that may serve as a binding site for adaptor modules like SH3 domains. Kidins220 C-terminal half is very abundant in phosphorylatable residues, serine, threonine, and tyrosine, that could constitute docking sites for Ser/Thr binding domain-containing proteins or phosphotyrosine binding modules such as SH2 domains. It also bears a sterile-␣ motif or SAM domain (4) and a potential PSD95/SAP90, DGL/ZO-1 (PDZ) binding motif at the very C terminus (5), both candidates for protein-protein interactions.Kidins220 was cloned as the first physiological substrate for protein kinase D (PKD) (1). This kinase, also known as PKD1 or protein kinase C (PKC) (6, 7), belongs to a novel family of diacylglycerol (DAG)-stimulated Ser/Thr kinases distantly related to the PKC family, characterized by unique enzymatic properties and domain architecture (for a recent review, see Refs. 8 and 9). PKD has multiple domains, including two cysteine-rich repeats that constitute a C1 domain...

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-g -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.