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...

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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...

In order for neurons to perform their function, they must establish a highly polarized morphology characterized, in most of the cases, by a single axon and multiple dendrites. Herein we find that the evolutionarily conserved protein Kidins220 (kinase D-interacting substrate of 220-kDa), also known as ARMS (ankyrin repeat-rich membrane spanning), a downstream effector of protein kinase D and neurotrophin and ephrin receptors, regulates the establishment of neuronal polarity and development of dendrites. Kidins220/ARMS gain and loss of function experiments render severe phenotypic changes in the processes extended by hippocampal neurons in culture. Although Kidins220/ARMS early overexpression hinders neuronal development, its down-regulation by RNA interference results in the appearance of multiple longer axon-like extensions as well as aberrant dendritic arbors. We also find that Kidins220/ARMS interacts with tubulin and microtubule-regulating molecules whose role in neuronal morphogenesis is well established (microtubule-associated proteins 1b, 1a, and 2 and two members of the stathmin family). Importantly, neurons where Kidins220/ARMS has been knocked down register changes in the phosphorylation activity of MAP1b and stathmins. Altogether, our results indicate that Kidins220/ARMS is a key modulator of the activity of microtubuleregulating proteins known to actively regulate neuronal morphogenesis and suggest a mechanism by which it contributes to control neuronal development.Neuronal differentiation comprises several steps, among which the acquirement of a polarized axon-dendrite phenotype, with the corresponding asymmetrical distribution of proteins, is crucial. The morphological changes, followed by a neuron in order to polarize and form a single axon and multiple dendrites, are triggered by signaling cascades evoked by both intracellular and extracellular cues (1-4).Embryonic hippocampal neurons in culture constitute a model to study the mechanisms governing the establishment of polarity (2, 5, 6). These neurons undergo clear morphological changes during in vitro polarization. First, neurons attach to the plate and form lamellipodia and filopodia (stage 1). After several hours, they extend several minor immature neurites of apparent equivalent nature (stage 2) until one of these minor processes extends rapidly and becomes the axon (stage 3). The remaining neurites develop into dendrites (stage 4), after which neurons become morphologically and functionally mature (stage 5) (5, 6).During the early events of the establishment of polarity in this model, differences in local actin polymerization among the immature neurites play a crucial role in axonal determination (5, 7). In a similar manner, microtubule dynamics influence neuronal polarization, because local microtubule stabilization in one neurite specifies an axonal fate (8). Other known regulators of neuronal polarity and axon specification include proteins involved in polarized trafficking (4, 9, 10). However, how these different molecules are linked to t...

Protein kinase D (PKD) is a family of serine/threonine kinases that can be activated by many stimuli via protein kinase C in a variety of cells. This is the first report where PKD activation and localization is studied in glial cells. Herein, we demonstrate that P2Y(2) and P2X7 receptor stimulation of primary rat cerebellar astrocytes rapidly increases PKD1/2 phosphorylation and activity. P2Y(2) receptor response evokes a PKD1/2 activation that is dependent on a pertussis toxin-insensitive G protein, phospholipase C (PLC)-mediated generation of diacylglycerol, and protein kinase C. This mechanism is similar to the one described for other G-protein coupled receptors. In contrast, the way the ionotropic P2X7 receptor activates PKD1/2 is significantly different. Importantly, this response is not dependent on calcium entry, but depends on the activity of several phospholipases, including phosphoinositide-phospholipase C (PI-PLC), phosphatidylcholine-phospholipase C (PC-PLC) and also phospholipase D (PLD). Immunoblot and confocal microscopy analysis show that PKD1/2 activation by nucleotides is transient. The active kinase first moves to and concentrates in certain plasma membrane domains. Then, phosphorylated-PKD1/2 translocates to intracellular vesicles, where it remains active. All together, our results open the perspective of PKD1/2 being involved in many physiological functions where nucleotides play important roles not only in astrocytes but in other cell types bearing these receptors.

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.

The taxonomy of ceratioid fishes is challenging and frequently based on a small number of female specimens described for each species. Twenty ceratioid specimens caught on Flemish Cap and Grand Bank (western North Atlantic), representing 12 species and six families: Ceratias holboelli and Cryptopsaras couesii (Ceratiidae); Himantolophus albinares (Himantolophidae); Melanocetus johnsonii (Melanocetidae); Lophodolos acanthognathus, Oneirodes eschrichtii, Dolopichthys karsteni, and Leptacanthichthys gracilispinis (Oneirodidae); Caulophryne polynema (Caulophrynidae); and Haplophryne mollis, Linophryne brevibarbata, and L. bicornis (Linophrynidae) were identified by examination of morphological characters. DNA barcode sequences, from the 5′ end of the COI mitochondrial gene, were developed for 18 specimens and compared with all ceratioid barcode sequences available in public repositories. The analyses extended the ranges of some quantitative traits for certain species, highlighted the possible existence of cryptic species in C. couesii with distinct ranges in the Atlantic and Pacific oceans, and indicated a close relationship between Bertella and Dolopichthys meriting further attention. The authoritative identification of the 18 voucher specimens made possible detection of erroneous identifications of some sequences extracted from the repositories and highlighting of taxonomic conflicts that should be the subject of future studies.

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