Lipid Raft Disruption Triggers Protein Kinase C and Src-dependent Protein Kinase D Activation and Kidins220 Phosphorylation in Neuronal Cells

Abstract

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...

Cholesterol-Dependent Kinase Activity Regulates Transmitter Release from Cerebellar Synapses

“…In MDCK cells M␤CD treatment activates PKA without increasing cAMP levels, possibly by disrupting inhibitory complexes localized to lipid rafts (Burgos et al, 2004). Activation of PKC and src in M␤CD-treated PC12 cells is also thought to be a consequence of alterations to localized lipid domain interactions (Cabrera-Poch et al, 2004). In addition to direct effects on kinase activity, the activity of PP2A phosphatase is sensitive to cholesterol depletion (Wang et al, 2005), therefore phosphatase inactivation may also contribute to an increase in protein phosphorylation following M␤CD treatment.…”

“…Cholesterol depletion is known to increase the activity of several staurosporine-sensitive kinases including PKA, PKC and src (Burgos et al, 2004;Cabrera-Poch et al, 2004). The mechanisms linking cholesterol levels to kinase activity are not fully understood.…”

“…Cholesterol depletion can lead to activation of several protein kinases that are known regulators of neurotransmitter release (Burgos et al, 2004;Cabrera-Poch et al, 2004). Functional cerebellar impairment is associated with impaired cholesterol trafficking in Niemann-Pick C1 disease so we have used a cerebellar culture model system to determine whether the activity of kinases regulating neurotransmitter release in the cerebellum is sensitive to changes in membrane cholesterol content.…”

Kidins220/ARMS Modulates the Activity of Microtubule-regulating Proteins and Controls Neuronal Polarity and Development

“…However, how these different molecules are linked to the extracellular cues that may modulate these processes in vivo, and how these diverse processes are coordinated to generate a polarized and mature neuron is only beginning to be understood. Kidins220 (kinase D-interacting substrate of 220 kDa), also known as ARMS (ankyrin repeat-rich membrane-spanning), is an integral membrane protein associated with lipid rafts, abundant in the developing nervous system and in highly plastic areas of the adult brain (11)(12)(13). This protein was first identified as a substrate for protein kinase D (PKD) 4 (12).…”

Protein kinase D: a new player among the signaling proteins that regulate functions in the nervous system

“…PKD1 phosphorylates the scaffold protein Kidins220 [55][56][57][58] . Kidins220 transportation from the TGN to the plasma membrane requires the autophosphorylation of PKD1 at Ser916.…”