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Genes to Cells (2007) 12, 929-939. doi:10.1111/j.1365-2443.2007.01101.x
© 2007 Blackwell Publishing or its licensors
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PX-RICS, a novel splicing variant of RICS, is a main isoform expressed during neural development

 Tomoatsu Hayashi1, Toshio Okabe2, Yukiko Nasu-Nishimura1, Fumika Sakaue1, Susumu Ohwada2, Ken Matsuura1, Tetsu Akiyama1 and Tsutomu Nakamura1,*

1 Laboratory of Molecular and Genetic Information, Institute for Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
2 Second Department of Surgery, Gunma University School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
In our previous study, we identified RICS, a novel ß-catenin-interacting protein with the GAP activity toward Cdc42 and Rac1, and found that RICS plays an important role in the regulation of neural functions, including postsynaptic NMDA signaling and neurite outgrowth. Here we report the characterization of an N-terminal splicing variant of RICS, termed PX-RICS, which has additional phox homology (PX) and src homology 3 (SH3) domains in its N-terminal region. The PX domain of PX-RICS interacted specifically with phosphatidylinositol 3-phosphate [PtdIns(3)P], PtdIns(4)P and PtdIns(5)P. Consistent with this binding affinity, PX-RICS was found to be localized at the endoplasmic reticulum (ER), Golgi and endosomes. We also found that wild-type PX-RICS possessed much lower GAP activity than RICS, whereas a mutant form of PX-RICS whose PX domain lacks the binding ability to phosphoinositides (PIs) exhibited the GAP activity comparable to that of RICS. However, PX-RICS and RICS exhibited similar inhibitory effects on neurite elongation of Neuro-2a cells. Furthermore, we demonstrate that PX-RICS is a main isoform expressed during neural development. Our results suggest that PX-RICS is involved in early brain development including extension of axons and dendrites, and postnatal remodeling and fine-tuning of neural circuits.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
The Rho family of small GTPases including RhoA, Rac1 and Cdc42 are critical regulators of the actin cytoskeleton in eukaryotic cells, and are implicated in various cellular functions (Hall 1998; Symons & Settlemam 2000). In response to extracellular stimuli, the Rho GTPases shuttle between a GDP-bound inactive state and a GTP-bound active state, thereby serving as binary molecular switches for downstream signaling. This reciprocal conversion of the Rho GTPase activity is facilitated by three classes of regulatory proteins: the guanine nucleotide exchange factors (GEFs), the Rho GTPase-activating proteins (GAPs) and the guanine nucleotide dissociation inhibitors (GDIs). In contrast to GEFs which promote the release of bound GDP and subsequent binding of GTP, GAPs enhance the intrinsic GTPase activity of the Rho GTPases, thus accelerating the return of the proteins to the inactive state (Sasaki & Takai 1998). In the neural system, RhoGAPs and their substrate Rho GTPases have been implicated in the regulation of multiple processes in the morphological development of neurons, including axonal growth and guidance, dendritic elaboration and formation of synapses (Threadgill et al. 1997; Albertinazzi et al. 1998; Lehmann et al. 1999; Ruchhoeft et al. 1999; Luo 2000). It has been shown that p190RhoGAP is crucially involved in semaphorin signaling via interaction with plexins, transmembrane receptors for semaphorins, and modulates growth, branching, guidance and fasciculation of axons. Furthermore, recent studies on p190RhoGAP-deficient mice demonstrated that p190RhoGAP plays a central role in fear memory formation in the lateral amygdala. The discovery of the RhoGAP oligopherenin-1, which is associated with X-linked mental retardation, further highlights the importance of RhoGAP in the neural function. These findings suggest that RhoGAPs contribute to not only fundamental processes of neural morphogenesis but also higher brain functions (Moon & Zheng 2003).

Phosphoinositides (PIs) are known to have an important regulatory role in diverse cellular functions, including signal transduction at the cell surface, regulation of membrane traffic, cytoskeleton and nuclear events, and the permeability and transport functions of the membranes (Di Paolo & De Camilli 2006). Seven species of PIs contained in eukaryotic cells are generated by reversible phosphorylation of phosphatidylinositol (PtdIns) at positions 3, 4 and 5 of its inositol ring. Each of the seven PIs has a unique, non-uniform subcellular distribution. For example, PtdIns(4,5)P2 is predominantly localized to the plasma membrane, PtdIns(4)P to the Golgi and PtdIns(3)P to the early endosome. A number of proteins that catalyze metabolism of PIs or cooperate with PIs contain modules with unique binding preference to specific PIs, including the pleckstrin homology (PH) domain, FYVE domain and phox homology (PX) domain. These PI-binding modules facilitate recruitment of the proteins to the proper membrane compartments.

We previously identified RICS, a novel ß-catenin-interacting protein with the GAP activity for Cdc42 and Rac1 (Okabe et al. 2003) [also designated Grit (Nakamura et al. 2002), p200 RhoGAP (Moon et al. 2003), p250RhoGAP (Nakazawa et al. 2003) and GCGAP (Zhao et al. 2003) by other groups]. RICS is expressed predominantly in brain, especially in cerebral cortex, amygdala, thalamus, caudate putamen and hippocampus. In neurons, RICS is localized to the postsynaptic density, where it forms a complex with ß-catenin, N-cadherin, N-methyl-D-aspartate (NMDA) receptors and postsynaptic density-95 (PSD-95). The GAP activity of RICS is suppressed when it is phosphorylated by Ca2+/calmodulin-dependent protein kinase II (CaMKII), a downstream effector of NMDA receptors. These findings raise the intriguing possibility that RICS is involved in the remodeling of local actin networks to cross-link synaptic adhesion and synaptic transmission under NMDA signaling. Furthermore, RICS was found to be localized at growth cones in cultured hippocampal neurons. Neurons derived from RICS-disrupted mice extend longer neurites than those from wild-type mice and this phenotype is rescued by exogenous expression of wild-type RICS, but not by the GAP activity-deficient mutant of RICS (Nasu-Nishimura et al. 2006). Thus we speculate that RICS is also involved in the neurite extension in its GAP activity-dependent manner. Here we report the identification of PX-RICS, a splicing variant of RICS with several structural characteristics distinct from RICS.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Molecular cloning of the PX-RICS cDNA

In an attempt to identify the exon–intron structure around the translational start site of the RICS gene, we found an expressed sequence tag (EST) clone whose nucleotide sequence in the 3' region is identical to that in the 5' region of the RICS gene. The 5' region of this EST clone encodes an open reading frame (ORF) that is not contained in the RICS gene. Thus, this EST clone appears to encode a novel splicing variant of RICS protein with an additional N-terminal structure. We isolated a human full-length cDNA of this RICS variant and designated it PX-RICS. Sequencing analysis revealed a composite sequence containing a 6261-bp long ORF that encodes a 230 kDa protein with 2087 amino acids. Its mouse and Xenopus orthologs contain 6486 and 5820-bp long ORF, encoding predicted proteins of 2162 and 1940 amino acids, respectively. Comparison of the nucleotide sequence of the human PX-RICS cDNA to the genomic sequence revealed that the isolated cDNA is indeed an alternative splicing isoform of RICS (Fig. 1A).


Figure 1
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  		Figure 1  Structure of PX-RICS. (A) Scheme showing the 5' genomic structure of the human RICS gene. Ten exons (P1–P10) encoding the N-terminal 334 amino acids of PX-RICS are followed by a single non-coding exon (R1) of RICS, which contains an in-frame stop codon preceding the RICS start codon. Exon P1–P10 and R1 are independently connected with exon R2, resulting in the generation of two alternatively spliced mRNAs encoding PX-RICS and RICS, respectively. The PX-RICS promoter region contains a TATA box near the transcription start site (TSS) and binding elements for transcription factors such as c-Rel, p300 and ROR{alpha} within a region between TSS and nucleotide-200. The RICS promoter region does not contain any typical TATA box but contains binding elements for transcription factors such as AML-1a, GATA-1 and Elk-1. (B) The predicted N-terminal amino acid sequence of PX-RICS is aligned with those of TC-GAP and CISK (PX domain). The PX domain is boxed and the SH3 domain is underlined. Conserved amino acids are shown against a black background. The first Met of RICS is enlarged and bolded. Consensus Tyr of the PX domain is indicated by an asterisk. (C) Schematic structures of PX-RICS and other RhoGAPs. Percentages indicate amino acid sequence identities to PX-RICS.

 
The N-terminal region of PX-RICS contains the PX and SH3 domains, which are followed by the amino acid sequence identical to that of RICS. The N-terminal half of PX-RICS is highly homologous to a recently isolated Rho-GTPase activating protein, TC-GAP (Fig. 1B) (Chiang et al. 2003). The PX, SH3 and GAP domains of PX-RICS share 49.6%, 69.8% and 70.4% identity with those of TC-GAP, respectively (Fig. 1C).

Expression of PX-RICS protein

We previously found that four different anti-RICS antibodies, which were raised against different epitopes of RICS, respectively, recognize a 250 kDa protein in addition to the 210 kDa RICS proteins. The nature of the 250 kDa protein, however, has been unknown. Because the calculated molecular weight of PX-RICS is 230 kDa, we assumed that the 250 kDa protein corresponds to PX-RICS. To identify the PX-RICS protein, we raised rabbit polyclonal antibody against its unique N-terminal portion (amino acids 53–112) so that the antibody specifically recognizes PX-RICS but not RICS. Immunoblotting analysis revealed that the antibody reacts specifically with PX-RICS that were generated by in vitro translation or exogenously expressed in 293T cells (Fig. 2A).


Figure 2
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  		Figure 2  Expression of PX-RICS protein. (A) Identification of PX-RICS protein. Recombinant PX-RICS generated by in vitro translation, PX-RICS exogenously expressed in 293T cells and endogenous PX-RICS in cell lines indicated were detected by immunoblotting with anti-PX-RICS antibody. (B) Anti-PX-RICS antibody recognizes a 250 kDa protein. Adult mouse brain lysates were subjected to immunoprecipitation with non-immune rabbit IgG, anti-RICS antibody or anti-PX-RICS antibody with (+Ag) or without antigen, followed by immunoblotting with anti-PX-RICS (upper panel) or anti-RICS (lower panel) antibody. Black arrowheads indicate PX-RICS and the white arrowhead indicates RICS. (C) PX-RICS is a splicing variant of RICS. MCF-7 cells were transfected with shRNA constructs as indicated and cell lysates were subjected to immunobolotting with anti-PX-RICS (upper panel) or anti-RICS (middle panel) antibody, respectively. Anti-{alpha}-tubulin antibody was used as a control (bottom panel). (D, E) Expression profile of PX-RICS and RICS in cultured cell lines (D) and adult mouse tissues (E). Lysates [20 µg for (D) and 30 µg for (E)] were subjected to immunoblotting analysis. {alpha}-Tubulin was used as a loading control. Black arrowheads indicate PX-RICS and the white arrowhead indicates RICS. (F) PX-RICS is associated with ß-catenin, PSD-95, N-cadherin and NR2B in vivo. Lysates prepared from mouse brain were subjected to immunoprecipitation with the antibodies indicated, fractionated by SDS-PAGE, and immunoblotted with the antibodies indicated. +Ag, antibodies were pre-incubated with antigens before use in immunoprecipitation.

 
When mouse brain lysates were immunoprecipitated with anti-RICS antibody followed by immunoblotting with anti-RICS antibody, a doublet of 250 and 210 kDa proteins were detected as we previously reported (Fig. 2B). On the other hand, immunoblotting analysis with anti-PX-RICS antibody detected only the 250 kDa protein. Furthermore, when brain lysates were subjected to immunoprecipitation with anti-PX-RICS antibody followed by immunoblotting with anti-RICS antibody, only the 250 kDa protein was detected. These results suggest that the 250 kDa protein recognized by anti-RICS antibody is PX-RICS.

To further confirm that these two proteins are derived from two alternative splicing variants, respectively, we generated short hairpin RNAs (shRNAs) designed to specifically suppress PX-RICS expression, shRNA-PX-RICS-1 and -2. Immunoblotting analysis revealed that expression of PX-RICS, but not RICS, is inhibited in MCF-7 cells transfected with shRNA-PX-RICS-1 or 2 (Fig. 2C). On the other hand, shRNA-RICS, which recognizes the sequence common to PX-RICS and RICS, inhibited the expression of both PX-RICS and RICS. These results suggest that PX-RICS is a splicing variant of RICS.

We next examined the expression of PX-RICS and RICS proteins in cultured cell lines. PX-RICS was ubiquitously expressed in all cell lines examined, whereas RICS was expressed in a subset of cell lines, DLD-1, MCF-7 and A431 (Fig. 2D). We also examined the expression of PX-RICS and RICS proteins in various adult mouse tissues (Fig. 2E). PX-RICS and RICS proteins were predominantly expressed in brain. However, unlike RICS, PX-RICS was also expressed at relatively low levels in lung, kidney and spleen.

PX-RICS is associated with ß-catenin, PSD-95, N-cadherin and NR2B in vivo

RICS is known to be associated with ß-catenin, PSD-95, N-cadherin and NR2B in vivo (Okabe et al. 2003). We therefore examined whether PX-RICS could interact with these proteins. When a lysate prepared from mouse brain was subjected to immunoprecipitation with anti-PX-RICS antibody followed by immunoblotting with anti-ß-catenin antibody, we found that ß-catenin co-immunoprecipitates with PX-RICS (Fig. 2F). Similarly, we found that PSD-95, N-cadherin, and NR2B co-precipitate with PX-RICS (Fig. 2F). Precipitations of these proteins were inhibited by incubation of anti-PX-RICS antibody with antigen used for immunization. Thus, PX-RICS is also associated with ß-catenin, PSD-95, N-cadherin and NR2B in living cells.

The PX domain of PX-RICS preferentially binds to PtdIns(3)P, PtdIns(4)P and PtdIns(5)P

Recent reports demonstrate that the PX domain interacts with specific phospholipids, and thereby affects the subcellular localization of the PX-domain containing proteins (Cullen et al. 2001; Seet & Hong 2006). To investigate whether the PX domain of PX-RICS would bind to specific phospholipids, we performed a protein-lipid overlay assay using a recombinant Myc-tagged PX domain (PX) and mutant PX domain (PX-Y173A), in which Tyr-173 was replaced with Ala. Tyr-173 corresponds to the conserved Tyr required for interaction with phospholipids (Virbasius et al. 2001; Seet & Hong 2006). As shown in Fig. 3B, PX strongly interacted with PtdIns(3)P, PtdIns(4)P and PtdIns(5)P, whereas PX-Y173A showed no specific interaction. A protein-lipid binding assay using PIP arrays also revealed that the PX domain of PX-RICS strongly interacted with PtdIns(4)P and PtdIns(5)P and moderately with PtdIns(3)P (Fig. 3C). To further confirm these results, we performed a liposome-binding assay (Lee et al. 2005). Consistent with the results from protein-lipid overlay assays, the PX domain of PX–RICS was co-precipitated most effectively with PtdIns(4)P- and PtdIns(5)P-containing vesicles and moderately with PtdIns(3)P-containing vesicles (Fig. 3D).


Figure 3
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  		Figure 3  The PX domain of PX-RICS preferentially binds to PtdIns(3)P, PtdIns(4)P and PtdIns(5)P. (A) Recombinant Myc-tagged PX domain used in protein-phospholipid binding assays (B–D). (B, C) The phospholipid-binding ability of Myc-tagged wild-type PX or PX-Y173A was analyzed by a protein-lipid overlay assay. PIP StripsTM and PIP arraysTM were incubated with wild-type PX or PX-Y173A (2 µg/mL) and proteins bound to lipids were detected with anti-Myc antibody. PE, phosphatidylethanolamine; PC, phosphatidylcholine; PS, phosphatidylserine; PA, phosphatidic acid; LPA, lysophosphatidic acid; LPC, lysophosphocholine; S1P, sphingosine-1-phosphate. (D) Liposome binding assay. Liposomes were composed of 66 µM phophatidylethanolamine, 33 µM phosphatidylcholine and 50 µM of indicated phospholipids. Then, Myc-tagged wild-type PX domain (100 ng) was added to liposomes and the mixture was incubated for 15 min. After centrifugation, the liposome pellets (P) and supernatants (S) were analyzed by SDS-PAGE followed by immunoblotting. The graph shows the percentage of PX domain bound to the liposome pellet containing PtdIns indicated. Error bars represent mean ± SEM of triplicate assays. 3P, PtdIns-3-P; 4P, PtdIns-4-P; 5P, PtdIns-5-P; 35P2, PtdIns-3, 5-P2; 45P2, PtdIns-4, 5-P2; 34P2, PtdIns-3, 4-P2; 345P3, PtdIns-3, 4, 5-P3. (E) COS-7 cells were transfected with wild-type PX or PX-Y173A fused to GFP. The PX domain (green), Golgi-marker GM130 (red) and nucleus (blue) were simultaneously detected by confocal microscopy. Bar, 10 µm.

 
Previous reports showed that PtdIns(3)P is mainly localized at the early endosome, PtdIns(4)P to the Golgi and endoplasmic reticulum (ER), and PtdIns(5)P in the nucleus (Behnia & Munro 2005; Di Paolo & De Camilli 2006). We therefore examined the localization of GFP-tagged wild-type and mutant PX domain of PX-RICS in COS-7 cells. The wild-type PX domain of PX-RICS was localized in the nucleus, Golgi and endosomes, whereas PX-Y173A diffusely distributed throughout the cytoplasm (Fig. 3E and data not shown). These results suggest that the PX domain of PX-RICS interacts with PtdIns(3)P, PtdIns(4)P and PtdIns(5)P in living cells.

PX-RICS localizes at the Golgi, ER and endosomes in NIH3T3 cells

We next examined the intracellular distribution of endogenous PX-RICS in NIH3T3 cells. The immunofluorescence signal for PX-RICS was observed as strong perinuclear punctuate staining and relatively less intense staining over the cytoplasm (Fig. 4A,E,I). Subsequently, we investigated the co-localization of endogenous PX-RICS with markers for the Golgi, ER and endosomes (Fig. 4B,F,J). When cells were stained with the combination of anti-PX-RICS antibody and the Golgi marker GM130, ER marker calnexin or early endosome marker Rab5, PX-RICS was partially co-localized with all of these markers (Fig. 4D,H,L). These results are consistent with the binding preference of the PX domain of PX-RICS to PtdIns(3)P, PtdIns(4)P and PtdIns(5)P.


Figure 4
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  		Figure 4  Localization of PX-RICS in NIH3T3 cells. NIH3T3 cells cultured for 24–36 h were double-labeled with antibodies to PX-RICS (A, E, I), GM130 (B), calnexin (F) or Rab5 (J). Nucleus was stained with TOPRO3 (C, G, K). Bars, 10 µm.

 
GAP activity of PX-RICS in vitro and in vivo

We have previously shown that RICS has RhoGAP activity toward Cdc42 and Rac1 (Okabe et al. 2003). To investigate if the N-terminal domain of PX-RICS affects its GAP activity, we performed in vitro and in vivo GAP assays. His-tagged RhoA was loaded with [{gamma}-32P] GTP and incubated in the presence or absence of Flag-tagged full-length wild-type PX-RICS or PX-RICS mutants indicated in Fig. 5A. Wild-type PX-RICS, PX-RICS-Y173A and wild-type RICS stimulated the GTPase activity of Cdc42 and Rac1, but had virtually no effect on RhoA. As expected, PX-RICS-R407M, in which essential Arg-407 in the catalytic site was replaced with Met, showed no activity toward any of the Rho GTPases examined. Interestingly, wild-type PX-RICS showed somewhat lower catalytic activity than RICS, whereas PX-RICS-Y173A, a mutant lacking lipid-binding ability, exhibited GAP activity comparable to that of RICS. In vivo RBD and PBD assays confirmed these results: wild-type PX-RICS possessed less GAP activity for Rac1 and Cdc42 than RICS or PX-RICS-Y173A (Fig. 5B,C). These results suggest that the GAP activity of PX-RICS may be regulated by the interaction with phosphatidylinositides via its PX domain.


Figure 5
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  		Figure 5  GAP activity of PX-RICS toward Rho GTPases. (A) In vitro GAP activity of full-length PX-RICS. Baculoviral His6-RhoA, -Cdc42 or -Rac1 were incubated in the presence or absence (Mock) of full-length wild-type PX-RICS, PX-RICS-R407M, PX-RICS-Y173A or wild-type RICS for the indicated times. (B) GAP activity of PX-RICS in HEK293T cells was analyzed by RBD and PBD assays. HEK293T cells were transfected with Myc-tagged RhoA, Cdc42 or Rac1 together with or without (Mock) full-length wild-type PX-RICS, PX-RICS-R407I, PX-RICS-Y173A or wild-type RICS. The amounts of the active forms of RhoA, cdc42 and Rac1 were assessed by a pull-down assay using GST-Rhotekin (RBD), which selectively binds GTP-bound RhoA, or GST-PAK3 (PBD), which selectively binds GTP-bound Cdc42 and Rac1. GST-RBD or -PBD precipitates were immunoblotted with anti-Myc antibody. (C) Quantification of the results of (B). Immunoblots were quantified using the NIH IMAGEJ software. Error bars represent mean ± SEM of triplicate assays.

 
PX-RICS is a main isoform expressed during neural development and regulates neurite outgrowth

We previously found that RICS is involved in axon elongation and NMDA signaling. We therefore compared the protein expression of RICS and PX-RICS during neural development. PX-RICS was detectable from embryonic day 13 and expressed strongly from embryonic day 15 onward (Fig. 6A). On the other hand, expression of RICS was detectable from postnatal 2 weeks. Immunoblotting of lysates from cultured hippocampal neurons prepared from embryonic day 16.5 mice showed similar expression patterns: PX-RICS was detectable from 1 day in vitro (DIV), whereas RICS was detectable from 14 DIV (data not shown). Immunofluorescent staining with anti-PX-RICS antibody revealed that PX-RICS is localized to the tip of a developing axon, and this is essentially the same staining pattern as that obtained with anti-RICS antibody (Fig. 6B). These results suggest that PX-RICS is a main isoform that plays an important role during embryonic and neural development.


Figure 6
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  		Figure 6  PX-RICS expression during neural development and neurite outgrowth. (A) Expression profiles of PX-RICS and RICS in mouse brain of various developmental stages. (B) Mouse primary hippocampal neurons cultured for 3 days were stained with anti-PX-RICS (B-a) or anti-RICS antibody (B-d). F-actin was labeled with Rhodamine-phalloidine (B-b, B-e). Growth cones are indicated with arrowheads. Bar, 5 µm. (C) Neuro-2a cells were transfected with empty vector (Mock) or Myc-tagged wild-type PX-RICS, PX-RICS-R407M or wild-type RICS, and were subjected to serum depletion for 48 h. The cells were stained with anti-Myc antibody. Bars, 10 µm. (D) Neurite lengths were measured using LSM 510 META software (Zeiss). Error bars represent mean ± SEM of triplicate assays.

 
We previously reported that hippocampal and cerebellar primary neurons from RICS KO mice bore longer neurites than those from wild-type mice. Thus, we examined the effect of exogenous PX-RICS on neurite outgrowth. The mouse neuroblastoma cell line Neuro–2A was transfected with PX-RICS or RICS and neurite length was measured after serum starvation for 48 h. As shown in Fig. 6C,D, wild-type PX-RICS and wild-type RICS exhibited the similar inhibitory effects on neurite extension, whereas PX-RICS-R407M showed no inhibitory effect. Thus, PX-RICS has a potential to regulate neurite extension.


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
We previously found that four different anti-RICS antibodies, which were raised against different epitopes of RICS, respectively, recognize a doublet of 210 and 250 kDa proteins by immunoblotting analysis (Okabe et al. 2003). RICS was believed to correspond to the 210 kDa protein, whereas the nature of the 250 kDa protein has been elusive (Nakamura et al. 2002; Moon et al. 2003; Nakazawa et al. 2003; Zhao et al. 2003). In this report, we have identified a novel splicing variant of RICS, termed PX-RICS, which corresponds to the 250 kDa protein.

The N-terminal region of PX-RICS contains a PX domain, one of the known PI-binding modules. We demonstrated that the PX domain of PX-RICS specifically binds to PtdIns(3)P, PtdIns(4)P and PtdIns(5)P. PtdIns(3)P is known to be predominantly distributed to the endosomes, PtdIns(4)P to the ER and Golgi and PtdIns(5)P in the nucleus. Consistent with this, endogenous PX-RICS in NIH3T3 cells was localized to the endosomes, Golgi and ER. Thus, the recruitment of PX-RICS to these membranous organelles may be ensured by the interaction with the phospholipids via its PX-domain.

RICS expression was mainly observed in brain but barely detectable in cell lines we examined, whereas PX-RICS was expressed in brain and several tissues including lung, liver and spleen, and all cell lines examined. Thus, RICS may regulate local actin dynamics through its GAP activity mainly in neurons as we have previously demonstrated, whereas PX-RICS may have more universal functions in multiple tissues. Several lines of evidence suggest that PX domain-containing proteins participate in diverse cellular functions, particularly in some aspects of membrane trafficking (Sato et al. 2001; Seet & Hong 2006). Thus it is interesting to speculate that PX-RICS might serve as a regulator of membrane trafficking via its PX domain.

In vitro and in vivo GAP assays revealed that the GAP activity of PX-RICS is low compared to that of RICS. Interestingly, PX-RICS-Y173A, a mutant unable to bind to PIs, showed the GAP activity comparable to RICS. Thus, the GAP activity of PX-RICS may be modulated by the interaction with PIs via its PX domain. The interaction of PX-RICS with PIs and perhaps some other proteins would alter the conformation of PX-RICS, thereby preventing its GAP domain from the interaction with Cdc42 and Rac1. However, the detailed molecular mechanisms of this negative regulation remain to be investigated.

Accumulating evidence suggest that RICS serves as a regulator of neurite outgrowth (Nakamura et al. 2002; Moon et al. 2003; Nakazawa et al. 2003; Zhao et al. 2003, Nasu-Nishimura et al. 2006). In an attempt to elucidate the functional difference between RICS and PX-RICS in the neural system, we first examined the expression patterns of PX-RICS and RICS proteins during embryonic and postnatal brain development. We found that PX-RICS is detectable by embryonic day 13, whereas RICS was detectable only after postnatal 2 weeks. It is therefore possible that PX-RICS is involved in early brain development including extension of axons and dendrites, and that RICS function is also required for postnatal remodeling and fine-tuning of neural circuits. Indeed, we found that PX-RICS has the ability to regulate neurite outgrowth in Neuro-2a cells. Elucidation of the molecular mechanism by which PX-RICS regulates neurite elongation is an important issue for future studies.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Plasmid construction

The human PX-RICS cDNA was obtained from a colon cancer cDNA library by PCR. Mammalian expression vectors were constructed in pcDNA3.1-Flag or pMKITNeo-Myc vector using standard methods. pEF-BOS-Myc RhoA, Cdc42 and Rac1 were provided by T. Tezuka. For baculovirus expression of PX-RICS, wild-type and mutant PX-RICS cDNA fragments encoding amino acids 117–255 (PX domain) were cloned in-flame into pFastBac HT{alpha}. PX-RICS cDNA sequence has been submitted to GENBANK database under accession no. EF127492.

Antibodies

Polyclonal antibody against PX-RICS was prepared by immunizing rabbits with human PX-RICS (amino acids 53–112) fused to a glutathione S-transferase (GST). The antibody was purified by affinity chromatography using a column to which the antigen used for immunization had been linked. Anti-RICS antibody was prepared as described previously (antigen: amino acids 1518–1578) (Okabe et al. 2003). Antibodies to GM130, calnexin and Rab5 (for immunocytochemistry), anti-ß-catenin, N-cadherin and NR2B (for immunoblotting) were purchased from BD Transduction laboratories. Anti-PSD-95 (for immunoblotting) was from Upstate Biotechnology. Anti-Myc 9E10 and anti-Flag M2 antibodies (for immunocytochemistry and for immunoblotting, respectively) were obtained from Santa Cruz Biotechnology and Sigma, respectively. Antibody to {alpha}-tubulin (for immunoblotting) was from Calbiochem. Rhodamine-Phalloidin (for staining F-actin) was purchased from Molecular Probes.

Cell culture and transfection

HEK293T, COS-7, MCF-7 and Neuro-2a cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS). NIH3T3 cells were cultured in DMEM supplemented with 10% calf serum. HEK293T, COS-7 and Neuro-2a cells were transfected using LipofectAMINE PLUS (Invitrogen). Hippocampal primary neuronal cultures were prepared basically as previously described (Okabe et al. 2003). Briefly, neurons from embryonic day 16.5 mouse embryos were plated on coverslips coated with poly-L-lysine (1 mg/mL) and grown in Neurobasal medium (Invitrogen) supplemented with B-27 supplement (Invitrogen) and 0.5 mM glutamine. After 3 days, cultured hippocampal neurons were fixed with 4% paraformaldehyde in phosphate-buffered saline for 10 min at room temperature and then ice-cold methanol for 10 min at –20 °C. Cells were double-stained with antibodies to PX-RICS or RICS and Rhodamine-Phalloidin.

Immunoprecipitation and immunoblotting

Immunoprecipitation and immunoblotting were performed as described previously (Okabe et al. 2003).

Immunostaining

Cells were plated on glass coverslips in six-well plates at 2.0 x 105 cells/well, fixed with 10% formalin in phosphate-buffered saline (PBS) for 15 min at room temperature and then permeabilized with ice-cold methanol for 10 min at –20 °C. Cells were blocked with 3% fetal bovine serum and then incubated with appropriate antibodies in 3% FBS/PBS for 90 min. The cells were incubated with secondary antibodies conjugated with Alexa Fluor 488 or 594 in 3%FBS/PBS for 90 min. For F-actin staining, RITC-phalloidin was used instead of Alexa Fluor 594-conjugated secondary antibody. The fluorescent images were obtained with a confocal imaging system (LSM 510 META; Carl Zeiss).

RNAi experiment

shRNA constructs were designed using SIDIRECT software. The following complementary oligonucleotides were annealed and inserted into the Hind III/Bgl II site of pSuper-retro vector: shRNA-PX-RICS-1, 5'-GCTATCGCTGGCAACAAGA-3'; shRNA-PX-RICS-2, 5’-GCTCATTACGTTCTTACGA-3' (targeted against nucleotides 670–688 and 1023–1041 of human PX-RICS); shRNA-RICS, 5'-GCTGCACAG CATTCATTGA-3' (targeted against nucleotides 125–143 of human RICS, which correspond to nucleotides 1171–1190 of human PX-RICS). MCF-7 cells were transfected using the Nucleofector kit R (amaxa) following the manufacturer's instruction. After 24 h of transfection, puromycin (2 µg/mL) was added to culture medium and cells were cultured for 48 h.

Protein-lipid overlay assay

Myc-tagged PX domain (Myc-PX or Myc-PX Y173A) was generated in Sf9 cells using the baculovirus expression system (Invitrogen) following the manufacturer's instruction. Protein-lipid overlay assays were performed using PIP stripsTM and PIP arraysTM (Echelon). Briefly, PIP stripsTM and PIP arraysTM were blocked in 3% fatty acid-free BSA in TBS-T (10 mM Tris–HCl, pH 8.0, 150 mM NaCl, 0.1% Tween 20) at room temperature for 1 h. The strips and arrays were then incubated with 2.0 µg/mL of Myc-PX or Myc-PX Y173A overnight in the dark at 4 °C. Then the strips and arrays were washed 3 times in TBS-T and incubated for 1 h with anti-Myc antibody (1 : 1000) at room temperature. The strips and arrays were then incubated with horseradish peroxidase-conjugated anti-mouse IgG antibody (1 : 15 000; GE Healthcare Biosciences) for 1 h at room temperature. The signals were detected by enhanced chemiluminescence (ECL) (GE Healthcare Biosciences).

Liposome-binding assay

Liposome binding assays were performed as described previously (Lee et al. 2005) with a slight modification. Briefly, phospholipid mixtures (66 µM phosphatidylethanolamine, 33 µM phosphatidylcholine, 50 µM phospholipids indicated in Fig. 3D) were dried, resuspended in 150 µL of buffer A (50 mM Hepes, pH 7.5, 3 mM MgCl2, 2 mM CaCl2, 3 mM EGTA, 80 mM KCl) and sonicated in a bath sonicator for 10 min. Purified Myc-PX (100 ng/reaction) was added and incubated at 37 °C for 15 min. After centrifugation for 30 min at 100 000 x g at 4 °C, pellets were resuspended in 150 µL of buffer A. The supernatants and pellets measuring 20 µL were subjected to SDS-PAGE followed by immunoblotting. The intensity of the stained bands was quantified using NIH Image J.

In vitro GAP assay

In vitro GAP assays were performed as described previously (Okabe et al. 2003). 293T cells were transfected with Flag-tagged full-length PX-RICS and cell lysates (200 µg of protein) were immunoprecipitated with anti-Flag M2 (1 µL) antibody immobilized on protein G-Sepharose beads. Immunoprecipitates were suspended in GAP buffer (20 mM Tris–Hcl, pH 7.5, 0.1 mM DDT, 1 mM GTP, 0.86 mg/mL BSA). His6-tagged Rho GTPases (0.1 µg of each protein) was pre-incubated in the presence of [{gamma}-32P] GTP (3 µCi) in a mixture containing 20 mM Tris–HCl, pH 7.5, 25 mM NaCl, 5 mM EDTA, 0.1 mM DDT for 10 min at 30 °C. After the addition of MgCl2 (final 20 mM), the Flag-PX-RICS immunoprecipitates described above were added to the reaction mixture (final volume 50 µL) and incubated at 30 °C. Samples (10 µL) were removed from the mixture at 0, 2, 4 and 6 min and diluted in 1 mL of ice-cold stop buffer (50 mM Tris–HCl, pH 7.5, 50 mM NaCl, 5 mM MgCl2). During sampling, the reaction mixture was gently suspended every 30 s. Samples were filtered through nitrocellulose membranes pre-wetted with stop buffer. After washing twice with 10 mL ice-cold stop buffer, the radioactivity remained on the filter was measured.

RBD and PBD assay

293T cells transfected with Myc-tagged RhoA, Cdc42 or Rac1 along with Flag-tagged wild-type or mutant PX-RICS were lysed with ice-cold lysis buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 10 mM MgCl2 1 mM PMSF, 10 µg/mL aprotinin, 10 µg/mL leupeptin, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate). The lysates were centrifuged at 17 400 x g for 35 min, and the supernatants were incubated for 30 min at 4 °C with 20 µg of GST-PBD (for Cdc42 and Rac1) or GST-RBD (for RhoA). GTP-bound RhoA, Cdc42 and Rac1 were detected by immunoblotting with anti-Myc antibody. The intensity of the stained bands was quantified using NIH Image J.


   Acknowledgements
 
Supported by Grants-in-Aid for Scientific Research on Priority Areas and the Organization for Pharmaceutical Safety and Research.


   Footnotes
 
Communicated by: Shigeo Koyasu

* Correspondence: E-mail: nakamura{at}iam.u-tokyo.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
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Received: 18 December 2006
Accepted: 1 May 2007




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