The polo-like protein kinases Fnk and Snk associate with a Ca2+- and integrin-binding protein and are regulated dynamically with synaptic plasticity
1999; Springer Nature; Volume: 18; Issue: 20 Linguagem: Inglês
10.1093/emboj/18.20.5528
ISSN1460-2075
Autores Tópico(s)RNA Research and Splicing
ResumoArticle15 October 1999free access The polo-like protein kinases Fnk and Snk associate with a Ca2+- and integrin-binding protein and are regulated dynamically with synaptic plasticity Gunther Kauselmann Gunther Kauselmann Present address: Artemis Pharmaceuticals GmbH, Neurather Ring 1, D-51063 Cologne, Germany Search for more papers by this author Markus Weiler Markus Weiler Zentrum für Molekulare Neurobiologie (ZMNH), University of Hamburg, Martinistraße 52, D-20246 Hamburg, Germany Search for more papers by this author Peer Wulff Peer Wulff Zentrum für Molekulare Neurobiologie (ZMNH), University of Hamburg, Martinistraße 52, D-20246 Hamburg, Germany Search for more papers by this author Sebastian Jessberger Sebastian Jessberger Zentrum für Molekulare Neurobiologie (ZMNH), University of Hamburg, Martinistraße 52, D-20246 Hamburg, Germany Search for more papers by this author Uwe Konietzko Uwe Konietzko Present address: The Salk Institute for Biological Studies, Laboratory of Genetics, 10010 North Torrey Pines Road, La Jolla, CA, 92037 USA Search for more papers by this author Joey Scafidi Joey Scafidi Center for Neural Science, New York University, New York, NY, 10003 USA Search for more papers by this author Ursula Staubli Ursula Staubli Center for Neural Science, New York University, New York, NY, 10003 USA Search for more papers by this author Jürgen Bereiter-Hahn Jürgen Bereiter-Hahn Department of Biology, J.W.Goethe-Universität, Marie-Curie-Straße 9, D-60439 Frankfurt, Germany Search for more papers by this author Klaus Strebhardt Klaus Strebhardt Department of Obstetrics and Gynecology, J.W.Goethe-Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany Search for more papers by this author Dietmar Kuhl Corresponding Author Dietmar Kuhl Zentrum für Molekulare Neurobiologie (ZMNH), University of Hamburg, Martinistraße 52, D-20246 Hamburg, Germany Search for more papers by this author Gunther Kauselmann Gunther Kauselmann Present address: Artemis Pharmaceuticals GmbH, Neurather Ring 1, D-51063 Cologne, Germany Search for more papers by this author Markus Weiler Markus Weiler Zentrum für Molekulare Neurobiologie (ZMNH), University of Hamburg, Martinistraße 52, D-20246 Hamburg, Germany Search for more papers by this author Peer Wulff Peer Wulff Zentrum für Molekulare Neurobiologie (ZMNH), University of Hamburg, Martinistraße 52, D-20246 Hamburg, Germany Search for more papers by this author Sebastian Jessberger Sebastian Jessberger Zentrum für Molekulare Neurobiologie (ZMNH), University of Hamburg, Martinistraße 52, D-20246 Hamburg, Germany Search for more papers by this author Uwe Konietzko Uwe Konietzko Present address: The Salk Institute for Biological Studies, Laboratory of Genetics, 10010 North Torrey Pines Road, La Jolla, CA, 92037 USA Search for more papers by this author Joey Scafidi Joey Scafidi Center for Neural Science, New York University, New York, NY, 10003 USA Search for more papers by this author Ursula Staubli Ursula Staubli Center for Neural Science, New York University, New York, NY, 10003 USA Search for more papers by this author Jürgen Bereiter-Hahn Jürgen Bereiter-Hahn Department of Biology, J.W.Goethe-Universität, Marie-Curie-Straße 9, D-60439 Frankfurt, Germany Search for more papers by this author Klaus Strebhardt Klaus Strebhardt Department of Obstetrics and Gynecology, J.W.Goethe-Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany Search for more papers by this author Dietmar Kuhl Corresponding Author Dietmar Kuhl Zentrum für Molekulare Neurobiologie (ZMNH), University of Hamburg, Martinistraße 52, D-20246 Hamburg, Germany Search for more papers by this author Author Information Gunther Kauselmann2, Markus Weiler1, Peer Wulff1, Sebastian Jessberger1, Uwe Konietzko3, Joey Scafidi4, Ursula Staubli4, Jürgen Bereiter-Hahn5, Klaus Strebhardt6 and Dietmar Kuhl 1 1Zentrum für Molekulare Neurobiologie (ZMNH), University of Hamburg, Martinistraße 52, D-20246 Hamburg, Germany 2Present address: Artemis Pharmaceuticals GmbH, Neurather Ring 1, D-51063 Cologne, Germany 3Present address: The Salk Institute for Biological Studies, Laboratory of Genetics, 10010 North Torrey Pines Road, La Jolla, CA, 92037 USA 4Center for Neural Science, New York University, New York, NY, 10003 USA 5Department of Biology, J.W.Goethe-Universität, Marie-Curie-Straße 9, D-60439 Frankfurt, Germany 6Department of Obstetrics and Gynecology, J.W.Goethe-Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany ‡G.Kauselmann and M.Weiler contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (1999)18:5528-5539https://doi.org/10.1093/emboj/18.20.5528 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info In order to stabilize changes in synaptic strength, neurons activate a program of gene expression that results in alterations of their molecular composition and structure. Here we demonstrate that Fnk and Snk, two members of the polo family of cell cycle associated kinases, are co-opted by the brain to serve in this program. Stimuli that produce synaptic plasticity, including those that evoke long-term potentiation (LTP), dramatically increase levels of both kinase mRNAs. Induced Fnk and Snk proteins are targeted to the dendrites of activated neurons, suggesting that they mediate phosphorylation of proteins in this compartment. Moreover, a conserved C-terminal domain in these kinases is shown to interact specifically with Cib, a Ca2+- and integrin-binding protein. Together, these studies suggest a novel signal transduction mechanism in the stabilization of long-term synaptic plasticity. Introduction Activity-dependent alterations in synaptic efficacy are thought to underlie learning and memory, epileptogenesis, drug abuse and several neurological diseases (Nestler and Aghajanian, 1997; Kuhl and Skehel, 1998; Milner et al., 1998). A primary cellular model for such synaptic plasticity is long-term potentiation (LTP; Bliss and Collingridge, 1993). Like memory, LTP can exist in both short- and long-lived forms. Short-lived forms of LTP rely on phosphorylation-dependent modifications of pre-existing proteins. The activity of both serine/threonine- and tyrosine-specific protein kinases has been implicated in this process (Grant, 1994; Grant and Silva, 1994; Huang et al., 1996; Roberson et al., 1996). Moreover, it has been proposed that post-translational modifications altering the activity of kinases and kinase-regulating proteins may act in the transfer of a short-lived LTP to longer lasting potentiations (Schwartz, 1993; Lisman, 1994). However, enduring forms of LTP require alterations in the molecular composition and structure of neurons, and are dependent on RNA and protein synthesis (Goelet et al., 1986; Curran and Morgan, 1987; Sheng and Greenberg, 1990; Kuhl, 1999). To understand the underlying genetic program, it will be necessary to identify the specific gene products that are increased in hippocampal neurons by plasticity-inducing neuronal activity. Currently, we know that the genomic response of synaptically activated neurons includes the induction of transcription factors (Morgan et al., 1987; Saffen et al., 1988), as well as proteins that may directly modify synaptic function (Nedivi et al., 1993; Qian et al., 1993; Yamagata et al., 1993; Link et al., 1995; Lyford et al., 1995; Frey et al., 1996; Brakeman et al., 1997). To this latter class of proteins belongs the serine/threonine kinase Pim-1 which we discovered previously in a subtractive screen for activity-dependent genes (Konietzko and Kuhl, 1998; Konietzko et al., 1999). Pim-1 is induced rapidly by plasticity-producing stimulation and is instrumental in the formation of enduring hippocampal LTP (Konietzko et al., 1999). The polo-like kinases are a family of serine/threonine-specific protein kinases that, like Pim-1, are induced as immediate early genes in non-neuronal cells (Glover et al., 1998; Nigg, 1998). Guided by this observation and the concept that certain aspects of cell cycle regulation and differentiation might be co-opted by the brain to serve functional plasticity, we examined here the influence of neuronal activity on the expression of the polo-like kinases in brain. In mammals, this family consists of three members, Plk, Fnk and Snk. Whereas the function of Snk is less clear, Plk and Fnk have been implicated in the control of multiple stages of cell division (Glover et al., 1998; Nigg, 1998). Our studies suggest a role for Fnk and Snk outside the cell cycle. Whereas we do not detect expression of Plk in the brain, both Fnk and Snk are constitutively expressed in post-mitotic neurons. Moreover, stimuli that induce seizures or LTP result in a dramatic increase in the synthesis of Fnk and Snk mRNA. This increase is reflected in a concomitant increase of Fnk and Snk protein in somata and dendrites of activated neurons. Both kinases interact with Cib, a protein previously shown to bind to Ca2+ and the cytoplasmic tail of integrin αIIb (Naik et al., 1997). The interaction of Fnk and Snk with Cib is dependent on the presence of the polo-box, a conserved C-terminal domain in the kinase proteins whose function has not been determined. The association of Fnk and Snk with an integrin-binding protein suggests a specific role in the stabilization of LTP. Results The family of polo-like kinases Three family members of the polo-like kinases, Plk, Fnk and Snk, have been described in mammals (Glover et al., 1998; Nigg, 1998). In addition, the human Prk gene was cloned and classified as the fourth member of this family (Li et al., 1996). All members of this family are characterized by the same domain topology, and alignment in a phylogenetic tree indicated that the polo-like kinases diverged before the subfamily of calmodulin kinase-related genes developed. As the first step of the present analysis, we cloned Fnk and Snk from rat brain (DDBJ/EMBL/GenBank accession Nos AF136584 and AF136583). Comparison of the deduced amino acid sequences of human Prk, rat Fnk and mouse Fnk (Donohue et al., 1995) shows that the three polypeptides are ∼90% identical except for a 17-amino-acid insertion present in rat Fnk and human Prk. However, Prk is lacking the most 5′ sequences that constitute the N-terminus of the predicted rodent proteins. As this is a very GC-rich sequence stretch and consequently difficult to extend for processing enzymes used in cloning, we suggest that Prk represents a truncated human sequence of Fnk (see also Chase et al., 1998). Moreover, sequences encoding the 17-amino-acid insertion of human and rat Fnk are also present in mouse mRNA from NIH-3T3 fibroblasts (DDBJ/EMBL/GenBank accession No. AF136586) and are flanked by two alternative 5′ consensus splice sites in the mouse genomic sequence. This suggests that the published sequence of mouse Fnk most likely represents a splice variant. Rat Fnk shares ∼50% sequence identity with rat Snk which is ∼90% identical to the mouse homolog (Simmons et al., 1992). The N-terminal half of Fnk and Snk harbors a serine/threonine-specific kinase domain including all 11 subdomains described to be specific for serine/threonine kinases (Hanks and Hunter, 1995). The C-terminal half contains a 30-amino-acid domain referred to as the polo-box which is highly conserved in all family members (Figure 1). This motif has not been described in any other protein and its function has not been determined. Figure 1.Comparison of C-terminal amino acid sequences of rat Fnk and Snk. Amino acid residues of Fnk (393–556) and Snk (412–590) are aligned. Identical amino acids are highlighted in blue. The polo-box (Polo30) and a larger region (Polo70) used in the two-hybrid analyses are framed and shown in dark and light gray, respectively. The proteins share 76.6% sequence identity in the polo-box region (Polo30). Download figure Download PowerPoint Levels of Fnk and Snk mRNA are regulated by neuronal activity Expression of Fnk and Snk in the brain of untreated rats and rats that had undergone a pentylenetetrazole (PTZ)-induced seizure was assayed by Northern blot analysis (Figure 2). The corresponding transcripts had a size of 2.4 (Fnk) and 2.9 (Snk) kb. Constitutive expression of Fnk mRNA was low but was induced 1.6-fold by seizure activity (Figure 2A and B). By comparison, basal expression of Snk was higher but was induced similarly by seizures. Induction was independent of new protein synthesis as it occurred in the presence of the protein synthesis inhibitor cycloheximide (CHX) (Figure 2C). Using in situ hybridization, we examined further the distribution and time course of basal and seizure-induced Fnk and Snk mRNA expression. In agreement with the Northern blot analysis, we observed low constitutive levels of Fnk mRNA in the hippocampus and cortex (Figure 3A). Higher constitutive levels were observed for Snk mRNA. In tissue of control rats, Snk mRNA levels were high in layers II, III, IV and VI of the occipital, parietal and temporal cortex. Regions with detectable basal expression also included the dentate gyrus, hippocampus proper, medial habenula, amygdala and putamen (Figure 3B). Hybridization to white matter was essentially the same as in sense control tissue (not shown). Following a PTZ-induced seizure, Fnk was strongly induced in the dentate gyrus and to a lesser extent in fields CA1 and CA3 of the hippocampus (Figure 3C). Snk was induced more broadly, with increased mRNA levels observable in cortical layers, fields CA1 and CA3 of the hippocampus and in the dentate gyrus (Figure 3D). In contrast to the effects of PTZ, kainic acid (KA)-induced seizures develop more slowly and recur for several hours (Ben-Ari et al., 1981). This stronger seizure episode resulted in additional increases in Fnk mRNA levels in layer VI of the temporal cortex (Figure 3E); Snk mRNA levels were elevated further in neocortex, hippocampal CA2 stratum pyramidale, striatum, amygdala and subthalamus (Figure 3F). Neither PTZ nor KA affected Plk expression, which was undetectable in brain (not shown). Seizure effects on Fnk and Snk kinase expression were not influenced by adrenalectomy (not shown), thereby demonstrating that corticosterone released during the seizure episode (Sun et al., 1993) does not account for the changes in gene expression described here. Figure 2.Regulation of Fnk and Snk mRNA levels in the hippocampus. (A) Autoradiograph of Northern blot analysis of Fnk-specific transcripts. A 2 μg aliquot of poly(A)+ RNA was loaded per lane. The blot was hybridized to a probe specific for Fnk. Hybridization to a probe specific for GAPDH was used as a loading control. Lane C, mRNA from saline-injected animals. Lane 1, mRNA isolated 1 h after PTZ-induced seizures. (B) Quantification of Fnk Northern blots given in bar diagrams. Error bars indicate SEMs (n = 3). Abbreviations are as in (A). (C) Autoradiograph of Northern blot analysis of Snk-specific transcripts. A 5 μg aliquot of total hippocampal RNA was loaded per lane. The blot was hybridized to probes specific for Snk and GAPDH. Lane C, RNA from saline-injected animals. Lanes 1, 4 and 10, the numbers indicate the time in hours after the onset of PTZ-induced seizures. Lane C/P, RNA isolated 4 h after the onset of PTZ-induced seizures in the presence of CHX. Lane K4, RNA isolated 4 h after the onset of KA-induced seizures. Download figure Download PowerPoint Figure 3.Comparative analysis of brain Fnk and Snk mRNA levels before and after seizure. Coronal sections were analyzed for Fnk mRNA (A, C and E) and Snk mRNA (B, D and F) using in situ hybridization with gene-specific antisense probes. (A and B) Control rat; (C and D) rat sacrificed 4 h after PTZ-induced seizure; (E and F) rat sacrificed 4 h after KA-induced seizures. a, amygdala; c, cortex; CA1–3, fields CA1–3 of the hippocampus; dg, dentate gyrus; mhb, medial habenula; sth, subthalamic nucleus; VI, layer VI of the cortex. Download figure Download PowerPoint To determine the specificity of induction of Fnk and Snk mRNA by neuronal activity, we examined the effects of electrical stimulation and the induction of LTP. LTP can be induced at synapses within the hippocampus by high-frequency orthodromic stimulation (Bliss and Collingridge, 1993). Induction of LTP was accompanied by increases in Fnk and Snk mRNA levels (Figure 4). Granule cells of the adult hippocampus were stimulated synaptically by activating their major afferent projection from the entorhinal cortex using a chronically implanted stimulating electrode (Staubli and Scafidi, 1997). Stimulation of the perforant path at the intensity required to produce a population spike, when administered at low frequency (0.2 Hz), did not result in LTP or an increase in Fnk or Snk mRNA levels (Figure 4A, C and E). By contrast, when LTP was evoked in the granule cells by delivering the same intensity stimuli at high frequency (400 Hz), Fnk and Snk were induced consistently in the ipsilateral dentate gyrus (Figure 4B, D and F). Fnk and Snk mRNAs were induced in each of four rats sacrificed 1 h after the LTP stimulation. These results demonstrate that of the three mammalian Plk genes, Fnk and Snk were expressed in the brain in overlapping but distinct populations of neurons and are regulated similarly by seizures and by electrical stimulation. Figure 4.Induction of LTP induces Fnk and Snk mRNAs in dentate gyrus granule cells in freely moving rats. Coronal sections were assayed for Fnk and Snk mRNA using in situ hybridization with gene-specific antisense probes. (A and B) Superimposed field potentials before and 1 h after (A) low-frequency stimulation (LFS) and (B) high-frequency stimulation (HFS) showing the induction of LTP with the latter. (C and D) Fnk mRNA levels 1 h after unilateral application of (C) LFS or (D) HFS. (E and F) Snk mRNA levels 1 h after unilateral application of (E) LFS or (F) HFS. The scale bar in (A) and (B) is 5 mV/2 ms. Download figure Download PowerPoint Fnk and Snk proteins are induced rapidly and enriched in somata and dendrites of activated neurons We next determined if the strong induction of Fnk and Snk mRNA by neuronal activity results in a corresponding increase in Fnk and Snk protein levels. In the hippocampal formation of control rats, immunoreactivity for both kinase proteins was localized to the somata and, at low basal levels, in the dendritic layers of the hippocampus proper and the dentate gyrus (Figure 5A and B). The distribution of Fnk and Snk immunostaining reflected patterns of mRNA expression; however, the relative levels of expressed kinase proteins differed from the mRNA levels observed in in situ hybridization and Northern blot analyses. Specifically, Fnk protein was detected at higher levels than the corresponding mRNA. This is most likely due to different affinities of the antisera for the corresponding protein; alternatively, the translation efficacy of the two kinase mRNAs might differ. However, as observed in the analysis of mRNA, recurrent KA-induced seizures markedly increased the immunoreactivity of both kinases (Figure 5C and D). The increase was most pronounced in the dentate gyrus granule cell somata and dendrites (Figure 5E–H), and in the pyramidal cell somata of field CA1 and their dendrites in the stratum radiatum (Figure 5I–L). Figure 5.Fnk and Snk proteins are localized to neuronal somata and dendrites of stimulated hippocampal neurons. Sections through hippocampi of control and KA-stimulated rat brain (4 h survival) were analyzed for Fnk protein (A, C, E, F, I and J) or Snk protein (B, D, G, H, K and L) using affinity-purified antisera. (A and B) Control hippocampus, only weak Fnk (A) and Snk (B) immunoreactivity is detected within the dentate gyrus and fields CA1–3. (C and D) After KA-induced seizures, Fnk (C) and Snk (D) immunoreactivity is increased in the granular and molecular layer of the dentate gyrus and in region CA1 with prominent staining of the dendritic processes. (E–H) High-power views of granule cells of the dentate gyrus before (E and G) and after KA-induced seizures (F and H). (I–L) High-power views of hippocampal field CA1 before (I and K) and after (J and L) KA-induced seizures. Sections from the same animal incubated with a serum depleted of either Fnk (M) or Snk (N) antibodies had no staining. (O) Immunoblots demonstrating the specific binding of the antisera to the corresponding recombinant kinase protein and that no cross-reactivity was observed. Recombinant Fnk protein (100 ng) was reacted with immunodepleted Fnk-specific antisera (lane 1), Fnk-specific antisera (lane 2) and Snk-specific antisera (lane 6). Recombinant Snk protein (100 ng) was reacted with immunodepleted Snk-specific antisera (lane 4), Snk-specific antisera (lane 5) and Fnk-specific antisera (lane 3). CA1–3, hippocampal fields CA1–3; dg, dentate gyrus; g, granular cell layer; p, pyramidal cell layer; slm, stratum lacunosum moleculare; sm, stratum moleculare; so, stratum oriens; sr, stratum radiatum. Download figure Download PowerPoint The Ca2+- and integrin-binding protein Cib specifically interacts with Fnk and Snk Cib had previously been isolated in a two-hybrid screen as a Ca2+-binding protein that interacts with the cytoplasmic tail of the integrin αIIb (Naik et al., 1997) and, in adult brain, integrins are concentrated in regions of synaptic contact (Einheber et al., 1996; Bahr et al., 1997). Importantly, a database entry (O.Yuan, DDBJ/EMBL/GenBank accession No. U83236) suggested that Cib might interact with Snk. We found that Cib is expressed constitutively in the brain: Northern analysis of hippocampal RNA identified a single band of ∼900 bp (Figure 6A). Using in situ hybridization, we examined the distribution of Cib mRNA expression. We observed constitutive levels of Cib mRNA in the hippocampus and cortex (Figure 6C) that were unaffected by seizure activity (Figure 6D). We further determined whether Cib protein shows an expression similar to that observed for Fnk and Snk proteins. Figure 7 shows that Cib immunoreactivity is localized to the somata and in the dendritic layers of the hippocampus proper and the dentate gyrus. As Cib was expressed in the hippocampus with an identical subcellular distribution to Fnk and Snk (compare Figures 7 and 5), we next determined if Snk and Fnk can interact with Cib. We cloned rat Cib from brain (DDBJ/EMBL/GenBank accession No. AF136585) and conducted a yeast two-hybrid analysis. Full-length Snk and Fnk cDNAs were expressed as GAL4 activator fusion proteins, and their interaction with Cib expressed as a GAL4 DNA-binding domain fusion protein was tested. Yeast co-transformed with Cib and either kinase expressed β-galactosidase. No β-galactosidase activity was observed when either protein was expressed alone or in the presence of unrelated proteins that were expressed as GAL4 activation or DNA-binding domain fusions. These findings were quantified in liquid β-galactosidase assays and are shown in Figure 8A and B. To determine the specific domain in Snk and Fnk responsible for the interaction, various segments of Snk and Fnk were co-expressed with Cib. Interaction was only observed in the presence of C-terminal fragments of Snk or Fnk; moreover, no interaction with Cib was seen with N-terminal fragments that encode the kinase domain. Figure 8A and B shows that 30 amino acids of the polo-box of Snk or Fnk were sufficient to confer binding to Cib. These interactions were of a strength similar to that observed for Cib and the full-length kinase proteins, and for Snf4 and Snf1 frequently used as positive controls in yeast interaction studies (Fields and Song, 1989). Figure 6.Cib mRNA is expressed in rat brain. Autoradiograph of Northern blot analysis of RNA extracted from cortex (A) and in situ hybridizations of coronal sections with gene-specific sense (B) and antisense (C and D) probes for Cib. (A) A 2 μg aliquot of poly(A)+ RNA was loaded. The blot was hybridized to a probe specific for Cib. Size markers in kilobases are indicated on the left. (B and C) Control rat, (D) rat sacrified 4 h after PTZ-induced seizure. CA1–3, hippocampal fields CA1–3 of the hippocampus; dg, dentate gyrus; mhb, medial habenula. Download figure Download PowerPoint Figure 7.Cib protein is localized to neuronal somata and dendrites of hippocampal neurons. Sections through hippocampi of rat brain were analyzed for Cib protein using a monoclonal mouse anti-Cib antibody (A–C). Immunoreactivity is detected within the dentate gyrus and fields CA1–3 of hippocampus (A). (B and C) High-power views show immunoreactivity in the granular and molecular layer of the dentate gyrus (B) and region CA1 with prominent staining of the dendritic processes (C). CA1–3, hippocampal fields CA1–3; dg, dentate gyrus; g, granular cell layer; p, pyramidal cell layer; slm, stratum lacunosum moleculare; sm, stratum moleculare; so, stratum oriens; sr, stratum radiatum. Download figure Download PowerPoint Figure 8.Analysis of interactions between Snk and Cib, and Fnk and Cib. (A) Yeast two-hybrid interaction analysis for Snk and Cib. (B) Yeast two-hybrid interaction analysis for Fnk and Cib. Assays in (A) and (B) were performed in liquid culture and β-galactosidase activity was quantified. The enzymatic assays shown represent the average of three independent co-transformants. Interaction was only observed between Cib and either the full-length kinase proteins, C-terminal fragments or polo-box domains. No interaction was seen between full-length kinases and lamin C, between Cib and an N-terminal Snk fragment, and between Cib and trypsinogen which was used as an additional negative control. Snf4 and Snf1 served as positive controls. Cib, complete coding region of Cib; Fnk, complete coding region of Fnk; FnkCT, C-terminal 313 amino acids of Fnk; FnkPolo30, 30 amino acids of the polo-box of Fnk; FnkPolo70, 70 amino acids of the polo-box domain of Fnk; Snk, complete coding region of Snk; SnkCT, C-terminal 331 amino acids of Snk; SnkNT, N-terminal 352 amino acids of Snk; SnkPolo30, 30 amino acids of the polo-box of Snk; SnkPolo70, 70 amino acids of the polo-box domain of Snk. (C) In vitro binding analysis of the interaction between Snk and Cib, and between Fnk and Cib. Myc-tagged GST protein (GST), Myc-tagged Snk C-terminal fusion protein (GSTSnkCT) and Myc-tagged Fnk C-terminal fusion protein (GSTFnkCT) were bound to HA-tagged GST–Cib fusion protein. The amount of bound Cib was quantified using a monoclonal mouse anti-HA antibody and a secondary peroxidase-conjugated mouse antibody. Relative binding data were: GST, 100%; GSTFnkCT, 925%; GSTSnkCT, 1114%. Download figure Download PowerPoint To determine whether the interaction between Cib and the kinases was direct, we analyzed their interaction using in vitro binding assays. As shown in Figure 8C, the C-terminal polypeptides of Snk and Fnk interacted with bacterially expressed glutathione S-transferase (GST)–Cib protein; there were no interactions with GST alone. Further evidence for the interaction of Cib with Snk came from the analysis of their subcellular localization. COS cells as well as the neuronal cell line, Neuro2A (N2A), were transfected with expression plasmids encoding green fluorescent protein (GFP)–Snk fusion protein and hemagglutinin (HA)-tagged Cib. Figure 9 shows that in COS and N2A cells expressing only Snk, Snk was observed mainly in the cytoplasm (Figure 9A and G, respectively). In contrast, in the two cell lines transfected with Cib only, Cib was distributed within the cytoplasm but there were also significant levels in the nucleus (Figure 9B and H). In ∼10% of COS cells transfected with Cib alone, the protein was observed exclusively in the nucleus (Figure 9C). Following double transfections of Cib and Snk, expression of Cib became restricted to the cytoplasm in COS as well as N2A cells (Figure 9E and K) and the localization of Cib resembled that seen for Snk (Figure 9D and J). Superposition of Snk and Cib immunostaining patterns indicated that their cytoplasmic distributions are largely identical (Figure 9F and C). Thus, a significant portion of Snk and Cib proteins are found in the same compartment (similar results were obtained for Fnk and Cib; data not shown). The results are consistent with the notion that Cib binds to Snk, although it is likely that Cib has additional roles in the nucleus where Snk is absent, at least in these transfected cells. Figure 9.Snk and Cib show identical subcellular localization when expressed together. Optical sections of single- and double-transfected COS and N2A cells were analyzed using confocal microscopy for the expression of GFP–Snk (green) or HA-tagged Cib detected by a Cy3-labeled secondary antibody (red). (A) Single transfected COS cell expressing GFP–Snk fusion protein. (B) Single transfected COS cell expressing HA-tagged Cib fusion protein. (C) Single transfected COS cell expressing HA-tagged Cib fusion protein with exclusive nuclear localization. (D) Double transfected COS cell expressing GFP–Snk fusion protein and HA-tagged Cib fusion protein. The localization of GFP–Snk fusion protein is shown. (E) The same cell as in (D). The localization of Cib fusion protein is shown. (F) Superposition of images shown in (D) and (E). (G) Single transfected N2A cell expressing GFP–Snk fusion protein. (H) Single transfected N2A cell expressing HA-tagged Cib fusion protein. (I) Phase-contrast image of the field containing the Cib-expressing cell sh
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