scholarly journals Shootin1: a protein involved in the organization of an asymmetric signal for neuronal polarization

2006 ◽  
Vol 175 (1) ◽  
pp. 147-157 ◽  
Author(s):  
Michinori Toriyama ◽  
Tadayuki Shimada ◽  
Ki Bum Kim ◽  
Mari Mitsuba ◽  
Eiko Nomura ◽  
...  
Keyword(s):  
Rna Interference ◽  
Hippocampal Neurons ◽  
Kinase Activity ◽  
Growth Cones ◽  
Intracellular Signals ◽  
Phosphoinositide 3 Kinase ◽  
Neuronal Polarization ◽  
Novel Protein

Neurons have the remarkable ability to polarize even in symmetrical in vitro environments. Although recent studies have shown that asymmetric intracellular signals can induce neuronal polarization, it remains unclear how these polarized signals are organized without asymmetric cues. We describe a novel protein, named shootin1, that became up-regulated during polarization of hippocampal neurons and began fluctuating accumulation among multiple neurites. Eventually, shootin1 accumulated asymmetrically in a single neurite, which led to axon induction for polarization. Disturbing the asymmetric organization of shootin1 by excess shootin1 disrupted polarization, whereas repressing shootin1 expression inhibited polarization. Overexpression and RNA interference data suggest that shootin1 is required for spatially localized phosphoinositide-3-kinase activity. Shootin1 was transported anterogradely to the growth cones and diffused back to the soma; inhibiting this transport prevented its asymmetric accumulation in neurons. We propose that shootin1 is involved in the generation of internal asymmetric signals required for neuronal polarization.

scholarly journals Identification of a Novel Cortactin SH3 Domain-Binding Protein and Its Localization to Growth Cones of Cultured Neurons

1998 ◽  
Vol 18 (10) ◽  
pp. 5838-5851 ◽  
Author(s):  
Yunrui Du ◽  
Scott A. Weed ◽  
Wen-Cheng Xiong ◽  
Trudy D. Marshall ◽  
J. Thomas Parsons
Keyword(s):  
Hippocampal Neurons ◽  
Binding Protein ◽  
Consensus Sequence ◽  
Growth Cones ◽  
Sh3 Domain ◽  
Actin Binding ◽  
Sequence Motifs ◽  
Cortical Actin ◽  
Cytoskeleton Reorganization

ABSTRACT Cortactin is an actin-binding protein that contains several potential signaling motifs including a Src homology 3 (SH3) domain at the distal C terminus. Translocation of cortactin to specific cortical actin structures and hyperphosphorylation of cortactin on tyrosine have been associated with the cortical cytoskeleton reorganization induced by a variety of cellular stimuli. The function of cortactin in these processes is largely unknown in part due to the lack of information about cellular binding partners for cortactin. Here we report the identification of a novel cortactin-binding protein of approximately 180 kDa by yeast two-hybrid interaction screening. The interaction of cortactin with this 180-kDa protein was confirmed by both in vitro and in vivo methods, and the SH3 domain of cortactin was found to direct this interaction. Since this protein represents the first reported natural ligand for the cortactin SH3 domain, we designated it CortBP1 for cortactin-binding protein 1. CortBP1 contains two recognizable sequence motifs within its C-terminal region, including a consensus sequence for cortactin SH3 domain-binding peptides and a sterile alpha motif. Northern and Western blot analysis indicated that CortBP1 is expressed predominately in brain tissue. Immunofluorescence studies revealed colocalization of CortBP1 with cortactin and cortical actin filaments in lamellipodia and membrane ruffles in fibroblasts expressing CortBP1. Colocalization of endogenous CortBP1 and cortactin was also observed in growth cones of developing hippocampal neurons, implicating CortBP1 and cortactin in cytoskeleton reorganization during neurite outgrowth.

scholarly journals The microtubule affinity regulating kinase MARK4 promotes axoneme extension during early ciliogenesis

2013 ◽  
Vol 200 (4) ◽  
pp. 505-522 ◽  
Author(s):  
Stefanie Kuhns ◽  
Kerstin N. Schmidt ◽  
Jürgen Reymann ◽  
Daniel F. Gilbert ◽  
Annett Neuner ◽  
...  
Keyword(s):  
Rna Interference ◽  
Protein Kinases ◽  
Basal Body ◽  
Kinase Activity ◽  
Complete Removal ◽  
Cilia Formation ◽  
Mother Centriole ◽  
Functional Analyses ◽  
Novel Protein

Despite the critical contributions of cilia to embryonic development and human health, key regulators of cilia formation await identification. In this paper, a functional RNA interference–based screen linked 30 novel protein kinases with ciliogenesis. Of them, we have studied the role of the microtubule (MT)-associated protein/MT affinity regulating kinase 4 (MARK4) in depth. MARK4 associated with the basal body and ciliary axoneme in human and murine cell lines. Ultrastructural and functional analyses established that MARK4 kinase activity was required for initiation of axoneme extension. We identified the mother centriolar protein ODF2 as an interaction partner of MARK4 and showed that ODF2 localization to the centriole partially depended on MARK4. Our data indicated that, upon MARK4 or ODF2 knockdown, the ciliary program arrested before the complete removal of the CP110–Cep97 inhibitory complex from the mother centriole, suggesting that these proteins act at this level of axonemal extension. We propose that MARK4 is a critical positive regulator of early steps in ciliogenesis.

scholarly journals The Saccharomyces cerevisiae checkpoint gene BUB1 encodes a novel protein kinase.

1994 ◽  
Vol 14 (12) ◽  
pp. 8282-8291 ◽  
Author(s):  
B T Roberts ◽  
K A Farr ◽  
M A Hoyt
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Normal Cell ◽  
Kinase Activity ◽  
Cell Multiplication ◽  
In Vitro Experiments ◽  
Microtubule Inhibitor ◽  
Cycle Progression ◽  
Novel Protein

Normal cell multiplication requires that the events of mitosis occur in a carefully ordered fashion. Cells employ checkpoints to prevent cycle progression until some prerequisite step has been completed. To explore the mechanisms of checkpoint enforcement, we previously screened for mutants of Saccharomyces cerevisiae which are unable to recover from a transient treatment with a benzimidazole-related microtubule inhibitor because they fail to inhibit subsequent cell cycle steps. Two of the identified genes, BUB2 and BUB3, have been cloned and described (M. A. Hoyt, L. Totis, and B. T. Roberts, Cell 66:507-517, 1991). Here we present the characterization of the BUB1 gene and its product. Genetic evidence was obtained suggesting that Bub1 and Bub3 are mutually dependent for function, and immunoprecipitation experiments demonstrated a physical association between the two. Sequence analysis of BUB1 revealed a domain with similarity to protein kinases. In vitro experiments confirmed that Bub1 possesses kinase activity; Bub1 was able to autophosphorylate and to catalyze phosphorylation of Bub3. In addition, overproduced Bub1 was found to localize to the cell nucleus.

Early phosphoinositide 3-kinase activity is required for late activation of protein kinase Cε in platelet-derived-growth-factor-stimulated cells: evidence for signalling across a large temporal gap

2001 ◽  
Vol 358 (2) ◽  
pp. 281-285 ◽  
Author(s):  
Egle BALCIUNAITE ◽  
Andrius KAZLAUSKAS
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Recent Observation ◽  
Time Interval ◽  
Long Time ◽  
Subsequent Activation ◽  
Phase Interval ◽  
Phosphoinositide 3 Kinase

At least two signalling systems have the potential to contribute to the activation of protein kinase C (PKC) family members such as PKC∊. One of these is phosphoinositide 3-kinase (PI 3-kinase), whose lipid products activate PKC∊ in vitro and in living cells. The recent observation that there are multiple waves of PI 3-kinase and PKC∊ activity within the G0-to-S phase interval provides a new opportunity to investigate the relationship between these two signalling enzymes in vivo. We have assessed the relative importance of the early and late waves of PI 3-kinase activity for the corresponding waves of PKC∊ activity. Blocking the first phase of PI 3-kinase activity inhibited both early and late activation of PKC∊. In contrast, the second wave of PI 3-kinase activity was dispensable for late activation of PKC∊. These findings suggested that early PI 3-kinase activation induced a stable change in PKC∊, which predisposed it to subsequent activation by lipid cofactors. Indeed, partial proteolysis of PKC∊ indicated that early activation of PI 3-kinase led to a conformation change in PKC∊ that persisted as the activity of PKC∊ cycled. We propose a two-step hypothesis for the activation of PKC∊ in vivo. One step is stable and depends on PI 3-kinase, whereas the other is transient and may depend on the availability of lipid cofactors. Finally, these studies reveal that PI 3-kinase and PKC∊ are capable of communicating over a relatively long time interval and begin to elucidate the mechanism.

scholarly journals Mechanism of A-kinase-anchoring protein 79 (AKAP79) and protein kinase C interaction

1999 ◽  
Vol 343 (2) ◽  
pp. 443-452 ◽  
Author(s):  
Maree C. FAUX ◽  
Emily N. ROLLINS ◽  
Amelia S. EDWARDS ◽  
Lorene K. LANGEBERG ◽  
Alexandra C. NEWTON ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Protein Interactions ◽  
Hippocampal Neurons ◽  
Kinase Activity ◽  
Catalytic Core ◽  
Kinase C ◽  
Anchoring Protein ◽  
Arginine Residues

The A-kinase-anchoring protein AKAP79 co-ordinates the location of cAMP-dependent protein kinase, phosphatase 2B (PP2B/calcineurin) and protein kinase C (PKC) at postsynaptic sites in neurons. In this report we focus on the mechanism of interaction between AKAP79 and PKC. We show that neither lipid activators nor kinase activation are required for association with AKAP79. The anchoring protein binds and inhibits the conserved catalytic core of PKCβII. AKAP79 also associates with conventional, novel and atypical isoforms of PKC in vitro andin vivo, and immunofluorescence staining of rat hippocampal neurons demonstrates that the murine anchoring-protein homologue AKAP150 is co-distributed with PKCα/β, PKCε or PKCℓ. Binding of the AKAP79(31-52) peptide, which inhibits kinase activity, exposes the pseudosubstrate domain of PKCβII, allowing endoproteinase Arg-C proteolysis in the absence of kinase activators. Reciprocal experiments have identified two arginine residues at positions 39 and 40 that are essential for AKAP79(31-52) peptide inhibition of PKCβII. Likewise, the same mutations in the full-length anchoring protein reduced inhibition of PKCβII. Thus AKAP79 associates with multiple PKC isoforms through a mechanism involving protein-protein interactions at the catalytic core where binding of the anchoring protein inhibits kinase activity through displacement of the pseudosubstrate.

scholarly journals A tightly associated serine/threonine protein kinase regulates phosphoinositide 3-kinase activity.

1993 ◽  
Vol 13 (3) ◽  
pp. 1657-1665 ◽  
Author(s):  
C L Carpenter ◽  
K R Auger ◽  
B C Duckworth ◽  
W M Hou ◽  
B Schaffhausen ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Phosphatase ◽  
Kinase Activity ◽  
Protein Phosphatase 2A ◽  
T Antigen ◽  
Serine Kinase ◽  
Phosphatase 2A ◽  
Phosphoinositide 3 Kinase

We identified a serine/threonine protein kinase that is associated with and phosphorylates phosphoinositide 3-kinase (PtdIns 3-kinase). The serine kinase phosphorylates both the 85- and 110-kDa subunits of PtdIns 3-kinase and purifies with it from rat liver and immunoprecipitates with antibodies raised to the 85-kDa subunit. Tryptic phosphopeptide maps indicate that p85 from polyomavirus middle T-transformed cells is phosphorylated in vivo at three sites phosphorylated in vitro by the associated serine kinase. The 85-kDa subunit of PtdIns 3-kinase is phosphorylated in vitro on serine at a stoichiometry of approximately 1 mol of phosphate per mol of p85. This phosphorylation results in a three- to sevenfold decrease in PtdIns 3-kinase activity. Dephosphorylation with protein phosphatase 2A reverses the inhibition. This suggests that the association of protein phosphatase 2A with middle T antigen may function to activate PtdIns 3-kinase.

Contact guidance of CNS neurites on grooved quartz: influence of groove dimensions, neuronal age and cell type

1997 ◽  
Vol 110 (23) ◽  
pp. 2905-2913 ◽  
Author(s):  
A. Rajnicek ◽  
S. Britland ◽  
C. McCaig
Keyword(s):  
Hippocampal Neurons ◽  
Growth Cones ◽  
Companion Paper ◽  
Contact Guidance ◽  
Neuronal Polarity ◽  
Cell Type ◽  
Spinal Cord Neurons ◽  
In Vitro System ◽  
Guidance Information

We used an in vitro system that eliminates competing guidance cues found in embryos to determine whether substratum topography alone provides important neurite guidance information. Dissociated embryonic Xenopus spinal cord neurons and rat hippocampal neurons were grown on quartz etched with a series of parallel grooves. Xenopus neurites grew parallel to grooves as shallow as 14 nm and as narrow as 1 microm. Hippocampal neurites grew parallel to deep, wide grooves but perpendicular to shallow, narrow ones. Grooved substrata determined the sites at which neurites emerged from somas: Xenopus neurites sprouted from regions parallel to grooves but presumptive axons on rat hippocampal neurons emerged perpendicular to grooves and presumptive dendrites emerged parallel to them. Neurites grew faster in the favored direction of orientation and turned through large angles to align on grooves. The frequency of perpendicular alignment of hippocampal neurites depended on the age of the embryos from which neurons were isolated, suggesting that contact guidance is regulated in development. Collectively, the data indicate that substratum topography is a potent morphogenetic factor for developing CNS neurons and suggest that in addition to a role in pathfinding the geometry of the embryo assists in establishing neuronal polarity. In the companion paper (A. M. Rajnicek and C. D. McCaig (1997) J. Cell Sci. 110, 2915–2924) we explore the cellular mechanism for contact guidance of growth cones.

The p101 subunit of PI3Kγ restores activation by Gβ mutants deficient in stimulating p110γ

2012 ◽  
Vol 441 (3) ◽  
pp. 851-858 ◽  
Author(s):  
Aliaksei Shymanets ◽  
Mohammad R. Ahmadian ◽  
Katja T. Kössmeier ◽  
Reinhard Wetzker ◽  
Christian Harteneck ◽  
...  
Keyword(s):  
Protein Kinase ◽  
G Protein ◽  
Crucial Role ◽  
Kinase Activity ◽  
Lipid Vesicles ◽  
Lipid Kinase ◽  
Dimeric Protein ◽  
Phosphoinositide 3 Kinase

G-protein-regulated PI3Kγ (phosphoinositide 3-kinase γ) plays a crucial role in inflammatory and allergic processes. PI3Kγ, a dimeric protein formed by the non-catalytic p101 and catalytic p110γ subunits, is stimulated by receptor-released Gβγ complexes. We have demonstrated previously that Gβγ stimulates both monomeric p110γ and dimeric p110γ/p101 lipid kinase activity in vitro. In order to identify the Gβ residues responsible for the Gβγ–PI3Kγ interaction, we examined Gβ1 mutants for their ability to stimulate lipid and protein kinase activities and to recruit PI3Kγ to lipid vesicles. Our findings revealed different interaction profiles of Gβ residues interacting with p110γ or p110γ/p101. Moreover, p101 was able to rescue the stimulatory activity of Gβ1 mutants incapable of modulating monomeric p110γ. In addition to the known adaptor function of p101, in the present paper we show a novel regulatory role of p101 in the activation of PI3Kγ.

scholarly journals Regulation of Phosphoinositide 3-Kinase by Its Intrinsic Serine Kinase Activity In Vivo

2004 ◽  
Vol 24 (3) ◽  
pp. 966-975 ◽  
Author(s):  
Lazaros C. Foukas ◽  
Caroline A. Beeton ◽  
Jorgen Jensen ◽  
Wayne A. Phillips ◽  
Peter R. Shepherd
Keyword(s):  
Growth Factor ◽  
Kinase Activity ◽  
Catalytic Subunit ◽  
Protein Kinase Activity ◽  
Growth Factor Signaling ◽  
Serine Kinase ◽  
Lipid Kinase ◽  
Phosphoinositide 3 Kinase

ABSTRACT One potentially important mechanism for regulating class Ia phosphoinositide 3-kinase (PI 3-kinase) activity is autophosphorylation of the p85α adapter subunit on Ser608 by the intrinsic protein kinase activity of the p110 catalytic subunit, as this downregulates the lipid kinase activity in vitro. Here we investigate whether this phosphorylation can occur in vivo. We find that p110α phosphorylates p85α Ser608 in vivo with significant stoichiometry. However, p110β is far less efficient at phosphorylating p85α Ser608, identifying a potential difference in the mechanisms by which these two isoforms are regulated. The p85α Ser608 phosphorylation was increased by treatment with insulin, platelet-derived growth factor, and the phosphatase inhibitor okadaic acid. The functional effects of this phosphorylation are highlighted by mutation of Ser608, which results in reduced lipid kinase activity and reduced association of the p110α catalytic subunit with p85α. The importance of this phosphorylation was further highlighted by the finding that autophosphorylation on Ser608 was impaired, while lipid kinase activity was increased, in a p85α mutant recently discovered in human tumors. These results provide the first evidence that phosphorylation of Ser608 plays a role as a shutoff switch in growth factor signaling and contributes to the differences in functional properties of different PI 3-kinase isoforms in vivo.

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