Role of highly central residues of P-loop and it's flanking region in preserving the archetypal conformation of Walker A motif of diverse P-loop NTPases

P-loop NTPases represent a large and highly diverse protein family that is involved in variety of cellular functions. Walker A motif forms a typical arched conformation, necessary to accommodate the phosphate moiety of the nucleoside tri (or di-) phosphate in Ploop NTPases. The feature that maintains the ancient architecture of P-loop is unidentified and uncharacterized. Here, using a well established global network parameter, closeness centrality, we identify that Walker A and its flanking regions (N- and C-terminal) have high density of globally connected residue positions. We find that closeness centrality of these residue positions are conserved across common structural core of diverse domains of P-loop NTPase fold. Our results suggest the potential role of globally connected residues in maintaining the local conformation of P-loop.     

Analysis of P-Loop and its Flanking Region Subsequence of Diverse NTPases Reveals Evolutionary Selected Residues

The P-loop NTPases are involved in diverse cellular functions. Members of the P-loop NTPase superfamily are characterized by presence of a highly conserved sequence pattern GxxxxGKS/T, known as Walker A motif. This motif adopts an archetypal P-loop conformation which allows accommodation of the triphosphate moiety of a bound nucleotide. Despite the presence of Walker A as a common sequence motif, P-loop NTPases exhibit extreme sequence divergence which hampers their phylogenetic or evolutionary classification. Here, we show that P-loop and its flanking region subsequence (termed as “extended-WalkerA motif”) contain distinct signatures that can be utilized to classify NTPase domain of functionally diverse proteins. We find a clearly classified group of diverse NTPases of Conserved Domain Database such as G-proteins, Ylqf, RecA like, DExDc, AAA, CPT, NK, ABC transporter and NifH proteins.     

The ankyrin repeat-rich membrane spanning (ARMS)/Kidins220 scaffold protein is regulated by activity-dependent calpain proteolysis and modulates synaptic plasticity

The expression of forms of synaptic plasticity, such as the phenomenon of long-term potentiation, requires the activity-dependent regulation of synaptic proteins and synapse composition. Here we show that ARMS (ankyrin repeat-rich membrane spanning protein)/Kidins220, a transmembrane scaffold molecule and BDNF TrkB substrate, is significantly reduced in hippocampal neurons after potassium chloride depolarization. The activity-dependent proteolysis of ARMS/Kidins220 was found to occur through calpain, a calcium-activated protease. Moreover, hippocampal long-term potentiation in ARMS/Kidins220(+/-) mice was enhanced, and inhibition of calpain in these mice reversed these effects. These results provide an explanation for a role for the ARMS/Kidins220 protein in synaptic plasticity events and suggest that the levels of ARMS/Kidins220 can be regulated by neuronal activity and calpain action to influence synaptic function.     

Developmental and activity-dependent regulation of ARMS/Kidins220 in cultured rat hippocampal neurons

Neurotrophin activation of Trk receptors elicits diverse effects on neuronal survival, differentiation, and synaptic plasticity. One of the central questions is how specificity is encoded in neurotrophin receptor signaling and actions. A unique downstream protein is the Ankyrin-Repeat Rich Membrane Spanning (ARMS)/Kinase D-interacting substrate-220 kDa (Kidins220), a very abundant scaffold protein in the hippocampus. To determine the roles of ARMS/Kidins220 in hippocampal neurons, we have analyzed the effects of synaptic activity upon the regulation and distribution of ARMS/Kidins220. At early times in vitro (<7 DIV), synaptic activity was low and ARMS/Kidins220 levels were high. As synaptic activity and markers for synapse maturation, such as PSD-95, increased, ARMS/Kidins220 significantly decreased to a plateau by later times in vitro (>12 DIV). Immunocytochemistry showed ARMS/Kidins220 to be concentrated at the tips of growing processes in immature cultures, and more diffusely distributed in older cultures. To examine the apparent inverse relationship between activity and ARMS/Kidins220 levels, neuronal firing was manipulated pharmacologically. Chronic exposure to TTX increased ARMS/Kidins220 levels, whereas bicuculline caused the opposite effect. Moreover, using shRNA to decrease ARMS/Kidins220 levels produced a corresponding increase in synaptic activity. We find that ARMS/Kidins220 may function in neuronal development as an indicator and potentially as a homeostatic regulator of overall synaptic strength in hippocampal neurons.     

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

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 microtubule-regulating proteins known to actively regulate neuronal morphogenesis and suggest a mechanism by which it contributes to control neuronal development.     

Ankyrin Repeat‐rich Membrane Spanning/Kidins220 protein regulates dendritic branching and spine stability in vivo

The development of nervous system connectivity depends upon the arborization of dendritic fields and the stabilization of dendritic spine synapses. It is well established that neuronal activity and the neurotrophin BDNF modulate these correlated processes. However, the downstream mechanisms by which these extrinsic signals regulate dendritic development and spine stabilization are less well known. Here we report that a substrate of BDNF signaling, the Ankyrin Repeat‐rich Membrane Spanning (ARMS) protein or Kidins220, plays a critical role in the branching of cortical and hippocampal dendrites and in the turnover of cortical spines. In the barrel somatosensory cortex and the dentate gyrus, regions where ARMS/Kidins220 is highly expressed, no difference in the complexity of dendritic arbors was observed in 1‐month‐old adolescent ARMS/Kidins220^+/? mice compared to wild‐type littermates. However, at 3 months of age, young adult ARMS/Kidins220^+/? mice exhibited decreased dendritic complexity. This suggests that ARMS/Kidins220 does not play a significant role in the initial formation of dendrites but, rather, is involved in the refinement or stabilization of the arbors later in development. In addition, at 1 month of age, the rate of spine elimination was higher in ARMS/Kidins220^+/? mice than in wild‐type mice, suggesting that ARMS/Kidins220^+/? levels regulate spine stability. Taken together, these data suggest that ARMS/Kidins220 is important for the growth of dendritic arbors and spine stability during an activity‐ and BDNF‐dependent period of development. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009     

Ankyrin repeat-rich membrane spanning/Kidins220 protein interacts with mammalian Septin 5

Neurotrophin receptors utilize specific adaptor proteins to activate signaling pathways involved in various neuronal functions, such as neurite outgrowth and cytoskeletal remodeling. The Ankyrin-Repeat Rich Membrane Spanning (ARMS)/kinase D-interacting substrate-220 kDa (Kidins220) serves as a unique downstream adaptor protein of Trk receptor tyrosine kinases. To gain insight into the role of ARMS/Kidins220, a yeast two-hybrid screen of a rat dorsal root ganglion library was performed using the C-terminal region of ARMS/Kidins220 as bait. The screen identified a mammalian septin, Septin 5 (Sept5), as an interacting protein. Co-immunoprecipitation using lysates from transiently transfected HEK-293 cells revealed the specific interaction between ARMS/Kidins220 and Sept5. Endogenous ARMS/Kidins220 and Sept5 proteins were colocalized in primary hippocampal neurons and were also predominantly expressed at the plasma membrane and in the tips of growing neurites in nerve growth factor-treated PC12 cells. Mapping of Sept5 domains important for ARMS/Kidins220 binding revealed a highly conserved N-terminal region of Sept5. The direct interaction between ARMS/Kidins220 and Sept5 suggests a possible role of ARMS/Kidins220 as a functional link between neurotrophin receptors and septins to mediate neurotrophin-induced intracellular signaling events, such as neurite outgrowth and cytoskeletal remodeling.     

Ankyrin Repeat-Rich Membrane Spanning (ARMS)/Kidins220 Scaffold Protein Regulates Neuroblastoma Cell Proliferation through p21

Cell proliferation is tightly controlled by the cell-cycle regulatory proteins, primarily by cyclins and cyclin-dependent kinases (CDKs) in the G₁ phase. The ankyrin repeat-rich membrane spanning (ARMS) scaffold protein, also known as kinase D-interacting substrate of 220 kDa (Kidins 220), has been previously identified as a prominent downstream target of neurotrophin and ephrin receptors. Many studies have reported that ARMS/Kidins220 acts as a major signaling platform in organizing the signaling complex to regulate various cellular responses in the nervous and vascular systems. However, the role of ARMS/Kidins220 in cell proliferation and cell-cycle progression has never been investigated. Here we report that knockdown of ARMS/Kidins220 inhibits mouse neuroblastoma cell proliferation by inducing slowdown of cell cycle in the G₁ phase. This effect is mediated by the upregulation of a CDK inhibitor p21, which causes the decrease in cyclin D1 and CDK4 protein levels and subsequent reduction of pRb hyperphosphorylation. Our results suggest a new role of ARMS/Kidins220 as a signaling platform to regulate tumor cell proliferation in response to the extracellular stimuli.     

Kidins220/ARMS is transported by a kinesin-1-based mechanism likely to be involved in neuronal differentiation

Kinase D-interacting substrate of 220 kDa/ankyrin repeat-rich membrane spanning (Kidins220/ARMS) is a conserved membrane protein mainly expressed in brain and neuroendocrine cells, which is a downstream target of the signaling cascades initiated by neurotrophins and ephrins. We identified kinesin light chain 1 (KLC1) as a binding partner for Kidins220/ARMS by a yeast two-hybrid screen. The interaction between Kidins220/ARMS and the kinesin-1 motor complex was confirmed by glutathione S-transferase-pull-down and coimmunoprecipitation experiments. In addition, Kidins220/ARMS and kinesin-1 were shown to colocalize in nerve growth factor (NGF)-differentiated PC12 cells. Using Kidins220/ARMS and KLC1 mutants, we mapped the regions responsible for the binding to a short sequence of Kidins220/ARMS, termed KLC-interacting motif (KIM), which is sufficient for the interaction with KLC1. Optimal binding of KIM requires a region of KLC1 spanning both the tetratricopeptide repeats and the heptad repeats, previously not involved in cargo recognition. Overexpression of KIM in differentiating PC12 cells impairs the formation and transport of EGFP-Kidins220/ARMS carriers to the tips of growing neurites, leaving other kinesin-1 dependent processes unaffected. Furthermore, KIM overexpression interferes with the activation of the mitogen-activated protein kinase signaling and neurite outgrowth in NGF-treated PC12 cells. Our results suggest that Kidins220/ARMS-positive carriers undergo a kinesin-1-dependent transport linked to neurotrophin action.     

Identification of a switch in neurotrophin signaling by selective tyrosine phosphorylation

Neurotrophins, such as nerve growth factor and brain-derived neurotrophic factor, activate Trk receptor tyrosine kinases through receptor dimerization at the cell surface followed by autophosphorylation and recruitment of intracellular signaling molecules. The intracellular pathways used by neurotrophins share many common protein substrates that are used by other receptor tyrosine kinases (RTK), such as Shc, Grb2, FRS2, and phospholipase C-gamma. Here we describe a novel RTK mechanism that involves a 220-kilodalton membrane tetraspanning protein, ARMS/Kidins220, which is rapidly tyrosine phosphorylated in primary neurons after neurotrophin treatment. ARMS/Kidins220 undergoes multiple tyrosine phosphorylation events and also serine phosphorylation by protein kinase D. We have identified a single tyrosine (Tyr(1096)) phosphorylation event in ARMS/Kidins220 that plays a critical role in neurotrophin signaling. A reassembled complex of ARMS/Kidins220 and CrkL, an upstream component of the C3G-Rap1-MAP kinase cascade, is SH3-dependent. However, Tyr(1096) phosphorylation enables ARMS/Kidins220 to recruit CrkL through its SH2 domain, thereby freeing the CrkL SH3 domain to engage C3G for MAP kinase activation in a neurotrophin dependent manner. Accordingly, mutation of Tyr(1096) abolished CrkL interaction and sustained MAPK kinase activity, a response that is not normally observed in other RTKs. Therefore, Trk receptor signaling involves an inducible switch mechanism through an unconventional substrate that distinguishes neurotrophin action from other growth factor receptors.     

Kidins220/ARMS is an essential modulator of cardiovascular and nervous system development

The growth factor family of neurotrophins has major roles both inside and outside the nervous system. Here, we report a detailed histological analysis of key phenotypes generated by the ablation of the Kinase D interacting substrate of 220 kDa/Ankyrin repeat-rich membrane spanning (Kidins220/ARMS) protein, a membrane-anchored scaffold for the neurotrophin receptors Trk and p75^NTR. Kidins220 is important for heart development, as shown by the severe defects in the outflow tract and ventricle wall formation displayed by the Kidins220 mutant mice. Kidins220 is also important for peripheral nervous system development, as the loss of Kidins220 in vivo caused extensive apoptosis of DRGs and other sensory ganglia. Moreover, the neuronal-specific deletion of this protein leads to early postnatal death, showing that Kidins220 also has a critical function in the postnatal brain.     

Kidins220/ARMS is a novel modulator of short-term synaptic plasticity in hippocampal GABAergic neurons

Kidins220 (Kinase D interacting substrate of 220 kDa)/ARMS (Ankyrin Repeat-rich Membrane Spanning) is a scaffold protein highly expressed in the nervous system. Previous work on neurons with altered Kidins220/ARMS expression suggested that this protein plays multiple roles in synaptic function. In this study, we analyzed the effects of Kidins220/ARMS ablation on basal synaptic transmission and on a variety of short-term plasticity paradigms in both excitatory and inhibitory synapses using a recently described Kidins220 full knockout mouse. Hippocampal neuronal cultures prepared from embryonic Kidins220(-/-) (KO) and wild type (WT) littermates were used for whole-cell patch-clamp recordings of spontaneous and evoked synaptic activity. Whereas glutamatergic AMPA receptor-mediated responses were not significantly affected in KO neurons, specific differences were detected in evoked GABAergic transmission. The recovery from synaptic depression of inhibitory post-synaptic currents in WT cells showed biphasic kinetics, both in response to paired-pulse and long-lasting train stimulation, while in KO cells the respective slow components were strongly reduced. We demonstrate that the slow recovery from synaptic depression in WT cells is caused by a transient reduction of the vesicle release probability, which is absent in KO neurons. These results suggest that Kidins220/ARMS is not essential for basal synaptic transmission and various forms of short-term plasticity, but instead plays a novel role in the mechanisms regulating the recovery of synaptic strength in GABAergic synapses.     

Kidins220/ARMS interacts with Pdzrn3, a protein containing multiple binding domains

We report the identification of a novel partner of Kidins220/ARMS (Kinase D-interacting substrate of 220 kDa/Ankyrin Repeat-rich Membrane Spanning) an adaptor of neurotrophin receptors playing crucial roles during neurogenesis. Screening a phage display library of brain cDNA products we found that D. rerio Pdzrn3, a protein containing RING-finger and PDZ-domains, interacts with Kidins220/ARMS through its first PDZ-domain. Both zebrafish proteins share high homology with the corresponding mammalian proteins and both genes are developmentally expressed in neural districts where early neurogenesis occurs. The interaction was also confirmed by biochemical assays and by co-localization at the tips of growing neurites of PC12 cells induced with nerve growth factor.     

Functional Interaction between the Scaffold Protein Kidins220/ARMS and Neuronal Voltage-Gated Na+ Channels

Kidins220 (kinase D-interacting substrate of 220 kDa)/ankyrin repeat-rich membrane spanning (ARMS) acts as a signaling platform at the plasma membrane and is implicated in a multitude of neuronal functions, including the control of neuronal activity. Here, we used the Kidins220^−/− mouse model to study the effects of Kidins220 ablation on neuronal excitability. Multielectrode array recordings showed reduced evoked spiking activity in Kidins220^−/− hippocampal networks, which was compatible with the increased excitability of GABAergic neurons determined by current-clamp recordings. Spike waveform analysis further indicated an increased sodium conductance in this neuronal subpopulation. Kidins220 association with brain voltage-gated sodium channels was shown by co-immunoprecipitation experiments and Na⁺ current recordings in transfected HEK293 cells, which revealed dramatic alterations of kinetics and voltage dependence. Finally, an in silico interneuronal model incorporating the Kidins220-induced Na⁺ current alterations reproduced the firing phenotype observed in Kidins220^−/− neurons. These results identify Kidins220 as a novel modulator of Na_v channel activity, broadening our understanding of the molecular mechanisms regulating network excitability.     

Human OLA1 defines an ATPase subfamily in the Obg family of GTP-binding proteins

Purine nucleotide-binding proteins build the large family of P-loop GTPases and related ATPases, which perform essential functions in all kingdoms of life. The Obg family comprises a group of ancient GTPases belonging to the TRAFAC (for translation factors) class and can be subdivided into several distinct protein subfamilies. The founding member of one of these subfamilies is the bacterial P-loop NTPase YchF, which had so far been assumed to act as GTPase. We have biochemically characterized the human homologue of YchF and found that it binds and hydrolyzes ATP more efficiently than GTP. For this reason, we have termed the protein hOLA1, for human Obg-like ATPase 1. Further biochemical characterization of YchF proteins from different species revealed that ATPase activity is a general but previously missed feature of the YchF subfamily of Obg-like GTPases. To explain ATP specificity of hOLA1, we have solved the x-ray structure of hOLA1 bound to the nonhydrolyzable ATP analogue AMPPCP. Our structural data help to explain the altered nucleotide specificity of YchF homologues and identify the Ola1/YchF subfamily of the Obg-related NTPases as an exceptional example of a single protein subfamily, which has evolved altered nucleotide specificity within a distinct protein family of GTPases.     

Identification and cloning of Kidins220, a novel neuronal substrate of protein kinase D

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 co-immunoprecipitates 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.     

Kidins220/ARMS mediates the integration of the neurotrophin and VEGF pathways in the vascular and nervous systems

Signaling downstream of receptor tyrosine kinases controls cell differentiation and survival. How signals from different receptors are integrated is, however, still poorly understood. In this work, we have identified Kidins220 (Kinase D interacting substrate of 220 kDa)/ARMS (Ankyrin repeat-rich membrane spanning) as a main player in the modulation of neurotrophin and vascular endothelial growth factor (VEGF) signaling in vivo, and a primary determinant for neuronal and cardiovascular development. Kidins220(-/-) embryos die at late stages of gestation, and show extensive cell death in the central and peripheral nervous systems. Primary neurons from Kidins220(-/-) mice exhibit reduced responsiveness to brain-derived neurotrophic factor, in terms of activation of mitogen-activated protein kinase signaling, neurite outgrowth and potentiation of excitatory postsynaptic currents. In addition, mice lacking Kidins220 display striking cardiovascular abnormalities, possibly due to impaired VEGF signaling. In support of this hypothesis, we demonstrate that Kidins220 constitutively interacts with VEGFR2. These findings, together with the data presented in the accompanying paper, indicate that Kidins220 mediates the integration of several growth factor receptor pathways during development, and mediates the activation of distinct downstream cascades according to the location and timing of stimulation.