Mechanobiology June 26th - June 2nd 2016

Mechanobiology: June 26th  - June 2nd 2016

Summary

Mutations in CCM3/PDCD10 result in cerebral cavernous malformations (CCMs), a major cause of cerebral hemorrhage. Despite intense interest in CCMs, very little is known about the function of CCM3. Here, we report that CCM3 is located on the Golgi apparatus, forming a complex with proteins of the germinal center kinase III (GCKIII) family and GM130, a Golgi-resident protein. Cells depleted of CCM3 show a disassembled Golgi apparatus. Furthermore, in wound-healing assays, CCM3-depleted cells cannot reorient the Golgi and centrosome properly, and demonstrate impaired migration. Golgi disassembly after either depletion of CCM3 or dissociation of CCM3 from the GM130-GCKIII complex is the result of destabilization of GCKIII proteins and dephosphorylation of their substrate, 14-3-3ζ. Significantly, the phenotype induced by CCM3 depletion can be reverted by expression of wild-type CCM3, but not by disease-associated mutants. Our findings suggest that Golgi dysfunction and the ensuing abnormalities of cell orientation and migration resulting from CCM3 mutations contribute to CCM pathogenesis.

Introduction

Cerebral cavernous malformations (CCMs) affect over 0.5% of the population (Labauge et al., 2007). They are vascular lesions in the brain characterized by enlarged vessels (caverns) lined by endothelium, with no underlying smooth muscle or elastic tissue (Moriarity et al., 1999). Bleeding occurs frequently in these lesions, and can lead to headaches, seizures, focal neurological deficits and stroke (Moriarity et al., 1999; Zabramski et al., 1999).

The majority of CCMs are sporadic, but a significant minority have a genetic basis (Labauge et al., 2007). Three different CCM loci have been identified: CCM1/KRIT1 (Laberge-le Couteulx et al., 1999), CCM2/OSM (Liquori et al., 2003) and CCM3/PDCD10 (Bergametti et al., 2005). Mutations are inherited in heterozygosis and loss of the wild-type allele in endothelial cells induces the development of the malformations (Akers et al., 2009; Pagenstecher et al., 2009). CCM1 and CCM2 are important in endothelial cell biology and vascular development (Hogan et al., 2008; Kleaveland et al., 2009; Whitehead et al., 2004; Whitehead et al., 2009). Biochemically, CCM2 is known to balance the activities of the small G proteins RhoA and cdc42 (Whitehead et al., 2009), probably through directed degradation of RhoA (Crose et al., 2009), whereas both CCM1 and CCM2 have been shown to form a complex that binds to the type I transmembrane receptor heart of glass (Kleaveland et al., 2009).

CCM3 has also been found in the CCM1-CCM2 complex, albeit only when one of the partners is overexpressed (Hilder et al., 2007; Stahl et al., 2008). Likewise, CCM3 has been found complexed to overexpressed phosphoprotein phosphatase type 2A (Goudreault et al., 2008), although the functional consequences of this interaction are not known.

The germinal center kinase III (GCKIII) subfamily of proteins consists of SOK1 (also referred to as YSK1 and STK25), Mst3 (also known as STK24) and Mst4 in mammals (Pombo et al., 2007; Ling et al., 2008). They belong to the Ste20 family of proteins, a large group of kinases that are characterized by a high degree of homology in their catalytic domain (Dan et al., 2001). The GCKIII kinases are involved in two important cellular processes: modulation of cell death and proliferation (Dan et al., 2002; Huang et al., 2002; Nogueira et al., 2008; Zhou et al., 2009), and regulation of cell migration (Lu et al., 2006; Preisinger et al., 2004). Specifically, Mst4 and SOK1 have been shown to associate with the cis-Golgi matrix protein GM130, where they play a role in determining the proper localization and morphology of the Golgi complex. SOK1 is also important for the invasion of cells into type I collagen and for cell migration in wound-healing assays, whereas overexpression of Mst4 seems to oppose these actions (Preisinger et al., 2004).

CCM3 has been shown to bind to GCKIII kinases, both in yeast two-hybrid assays (Ma et al., 2007; Rual et al., 2005) and in mammalian cells upon overexpression (Goudreault et al., 2008; Ma et al., 2007; Voss et al., 2007). The reported functional and biochemical consequences of this interaction are the modulation of the ERK pathway (Ma et al., 2007) and the phosphorylation of CCM3 by SOK1 (Voss et al., 2007).

In this work, we show for the first time that CCM3 is located on the Golgi apparatus, forming a complex with a GCKIII protein and GM130. CCM3 is important to maintain normal GCKIII protein levels and phosphorylation of the SOK1 substrate, 14-3-3ζ. As a consequence, cells with silenced CCM3 are impaired in their orientation and migration capabilities. These results link Golgi dysfunction and abnormalities in cell polarity to the development of cerebral cavernomas.

Fig. 1.

CCM3 localizes to the Golgi apparatus. (A) SaOS2 cells were transfected with control or CCM3 siRNA. CCM3 (red) and Golgi 58K (a Golgi marker, green) were stained 96 hours later by immunofluorescence and visualized by confocal microscopic analysis. Nuclei were identified by Hoechst 33342 staining (blue). Scale bar: 10 μm. (B) Rat liver homogenate and rat liver Golgi membranes were analyzed by immunoblotting using anti-CCM3, anti-GM130 and anti-Golgi-58K (as Golgi markers), or anti-tubulin (as a cytosolic marker).

Results

CCM3 is located on the Golgi apparatus

Because of its relevance to cavernoma pathogenesis, we wanted to study the cell biological role of CCM3 and the consequences of its mutation. When we performed an immunofluorescence analysis of CCM3 in cultured cells, we saw that the immunoreactivity of CCM3 was concentrated in a spot near the nucleus, where it overlapped with the cis-Golgi protein Golgi 58K (Fig. 1A) and also with another cis-Golgi protein, GM130 (supplementary material Fig. S1). As a control for the specificity of the immunofluorescence, we transfected SaOS2 cells with a small interfering (si)RNA against CCM3 (Fig. 2A). Under these circumstances, the immunoreactivity of the CCM3 antibody diminished significantly, suggesting that the detected signal is indeed CCM3. This localization of CCM3 was unexpected, as it is at odds with its reported interaction with other CCM gene products, such as CCM1 and CCM2, which have been shown to form a complex at the cell periphery (Hilder et al., 2007). To confirm the location of CCM3 with a different technique, we purified Golgi membranes from rat liver. CCM3 was readily detectable in the purified Golgi membranes (Fig. 1B). The amount of CCM3 in purified Golgi membranes compared with the amount in whole tissue extracts was similar to the amount of the known Golgi protein Golgi 58K, but was clearly lower than that of GM130. This could be due to either incomplete localization of CCM3 on the Golgi apparatus or its dissociation from the Golgi membranes during purification.

CCM3 is important for cell migration, and for Golgi assembly and repositioning

We next asked what is the biological function of CCM3. Therefore, we downregulated CCM3 using RNAi. Transient transfection of CCM3 siRNA in SaOS2 cells consistently reduced CCM3 levels by more than 90% (Fig. 2A, left panel). To rule out non-specific effects of the siRNA, we also downregulated CCM3 by RNAi using a lentivirally encoded small hairpin (sh)RNA directed against the 3′ untranslated region (UTR) of CCM3 mRNA in SaOS2 cells (Fig. 2A, middle panel) and HeLa cells (Fig. 2A, right panel). In both cell types, the shRNA was even more effective than the synthetic siRNA in downregulating CCM3 protein levels.

We then asked what effect knockdown of CCM3 had on cell polarity and migration, because its SOK1 binding partner has been shown to regulate these events (Preisinger et al., 2004). To that end, we studied the effect of CCM3 knockdown on cell migration using a wound-healing assay. We found that cell migration was significantly impaired in cells depleted of CCM3 (Fig. 2B,C). This was true in both SaOS2 and HeLa cells (Fig. 2C), confirming that this is not a cell-specific effect.

The impaired migration correlated with the inability of cells depleted of CCM3 to reposition both the Golgi apparatus and the centrosome towards the leading edge of the wound when treated with serum. This was in distinct contrast to control siRNA-treated cells, which repositioned their Golgi apparatus and centrosome quite efficiently (Fig. 2D-G). Significantly, this was the case for SaOS2 cells transfected with siRNA or transduced with shRNA, and for HeLa cells, for which the effect was evident even 6 hours after initiation of the wound-healing assay (Fig. 2E for SaOS2 and supplementary material Fig. S2 for HeLa).

Golgi disassembly and reassembly are important for the repositioning of the organelle in wound-healing assays; the ERK pathway is believed to be important for the disassembly step (Bisel et al., 2008). As CCM3 has been reported to activate ERK in other cellular systems (Ma et al., 2007), we assessed ERK activity in CCM3-depleted cells. Unexpectedly, CCM3 depletion did not affect ERK activation by serum (Fig. 3A), suggesting that CCM3 regulates Golgi repositioning by means of an ERK-independent mechanism. Furthermore, in both serum-deprived and serum-stimulated cells, CCM3 knockdown increased the number of cells with a dispersed Golgi. This was irrespective of cell type or method of CCM3 knockdown employed (Fig. 3B,C), and was even apparent in normal proliferating cells (Fig. 4A,B). Furthermore, the cells with dispersed Golgi were the same as those that had undetectable CCM3 by immunofluorescence (supplementary material Fig. S3). The same Golgi phenotype was seen in HeLa cells (supplementary material Fig. S2), confirming that this is not a cell-specific phenomenon.

The dispersion seen after CCM3 knockdown could be due to the inability of the cell to reassemble the organelle after its disassembly. To test this hypothesis, we induced Golgi dispersion with the fungal metabolite Brefeldin A (BFA). BFA induced comparable dispersal of the Golgi apparatus in both control cells and cells depleted of CCM3. However, whereas a compact Golgi was discernible in most control cells within 90 minutes of washout of BFA, a high percentage of cells with reduced CCM3 showed a dispersed tubuloreticular pattern even 180 minutes after washout of BFA (Fig. 4A, quantified in Fig. 4B and in supplementary material Fig. S2D for HeLa cells). Again, impaired Golgi reassembly was observed in all cell types tested and with both strategies of CCM3 knockdown. Furthermore, the effects of RNAi on Golgi reassembly were dependent on CCM3 knockdown, because re-expressing CCM3 by transfection (Fig. 4C) rescued the effects of CCM3 shRNA on Golgi reassembly in both control cells and after BFA treatment (Fig. 4D). Likewise, expression of CCM3 also rescued both Golgi and centrosome orientation in the wound-healing assay (Fig. 4E for Golgi and data not shown for centrosome), and cell migration in the same assay (Fig. 4F).

Fig. 2.

CCM3 is important for Golgi and centrosome repositioning, and cell migration. (A) SaOS2 cells were transiently transfected with control or CCM3 siRNA (left panel). Stable cell populations with control or CCM3 shRNA in SaOS2 cells (middle panel) and HeLa cells (right panel) were obtained by lentiviral transduction followed by puromycin selection. Cell extracts were analysed by immunoblotting using anti-CCM3 antibody. Anti-tubulin was used as a loading control. (B) SaOS2 and HeLa cells stably transduced with control or CCM3 shRNA were subject to wound-healing assays. Representative images at 4× magnification for HeLa cells are shown 0 and 24 hours after wound healing. (C) Migration in wound-healing assays was quantified by measuring the number of pixels in the denuded area, and expressed as the denuded area at time 0 hours minus the denuded area at time 24 hours (for HeLa cells) or 48 hours (for SaOS2 cells). Three wells were counted per condition in each experiment. The average and standard deviation of three independent experiments are shown. *P<0.01. (D) SaOS2 confluent monolayers were serum-deprived overnight, wounded and treated with 10% serum for 90 minutes (serum) or left untreated (SD). The Golgi apparatus was labeled with anti-GM130 antibody and DNA with Hoechst 33342. Golgi was scored as oriented (+) if more than half of the GM130 staining was laid within a 90° angle facing the wound edge (at the upper part of the micrograph). Scale bar: 20 μm. (E) Quantification of Golgi orientation. An average of 100 cells was counted per condition in each experiment. Discontinuous lines mark basal levels expected for a random orientation of 25%. The average and standard deviation of three independent experiments are shown. *P<0.01. (F,G) Centrosome orientation was assessed under the same conditions and with the same criteria as for Golgi (see D,E), using an anti-γ-tubulin antibody for staining. *P<0.01.

Fig. 3.

Lack of CCM3 results in Golgi disassembly. In all cases, SaOS2 cells were serum starved overnight, and then treated with 10% serum for 90 minutes (serum) or left untreated (SD). (A) Extracts from cells stably transduced with control or CCM3 shRNA were analyzed by immunoblotting using anti-p-ERK. Total ERK1,2 (ERK) was used as a loading control. (B) SaOS2 cells were analyzed by immunofluorescence confocal microscopy. The Golgi apparatus was visualized with antibodies against GM130 and Golgi 58K, and DNA was stained with Hoechst 33342. The arrows point to cells with dispersed Golgi. Scale bar: 10 μm. The lower panels show the dispersed Golgi area of a single cell (boxed region) at a higher magnification. (C) Quantification of Golgi dispersion of SaOS2 cells after transient transfection of control or CCM3 siRNA, and in stable cell populations with control or CCM3 shRNA. An average of 300 cells was counted per condition in each experiment. The average and standard deviation of three independent experiments are shown. *P<0.01.

CCM3 forms a complex with the cis-Golgi GCKIII proteins and GM130

CCM3 has been found bound to the other CCM proteins, CCM2 and CCM1, at least upon overexpression (Hilder et al., 2007; Stahl et al., 2008; Voss et al., 2007), and depletion of CCM2 has been shown to affect cytoskeletal organization, probably through modulation of the small G proteins cdc42 and RhoA (Crose et al., 2009; Whitehead et al., 2009). Because small G proteins have been implicated in the regulation of centrosome relocation and directed cell migration (Nobes and Hall, 1999), the CCM3 phenotype could be mediated by their modulation. However, our results show that stress fiber formation was not affected in CCM3-depleted cells, either when serum deprived or when stimulated with serum. Moreover, when we measured the activity of cdc42 and RhoA in serum-deprived and serum-stimulated cells, we found no difference between control and CCM3-depleted cells (supplementary material Fig. S4). We concluded that the effect of CCM3 depletion was probably mediated by a mechanism other than modulation of small G proteins.

The localization of CCM3 on the Golgi is probably caused by its binding to a protein or proteins present there. CCM3 has been reported to interact with several different proteins and protein complexes, among them the GCKIII family of kinases (Mst4, Mst3 and SOK1) (Goudreault et al., 2008; Ma et al., 2007; Rual et al., 2005; Voss et al., 2007). Two GCKIII family members (Mst4 and SOK1, but not Mst3) have been found in the cis-Golgi network, and SOK1 has been shown to be involved in Golgi repositioning and cell migration (Preisinger et al., 2004). We found that all three GCKIII proteins are present in purified Golgi membranes. Surprisingly, Mst4 was less enriched than the other two proteins; this correlated with reduced binding of Mst4 to the Golgi protein GM130 (supplementary material Fig. S5). We confirmed that CCM3 and the GCKIII kinase SOK1 interact in mammalian cells when overexpressed (Fig. 5A). Moreover, Mst3 and Mst4 also bind to CCM3 (Fig. 5B). The interaction of CCM3 with SOK1, Mst3 or Mst4 is not an artifact of overexpression, because we detected endogenous CCM3 in immunoprecipitates of endogenous SOK1 and Mst4 (Fig. 5C). Furthermore, although endogenous CCM3 could not be detected in immunoprecipitates of Mst3, Mst3 was present in CCM3 immunoprecipitates (Fig. 5D). To identify the domain of SOK1 important for interaction with CCM3, we co-transfected CCM3 with full-length SOK1, the N-terminal kinase domain (SOK1ΔC) or the C-terminal regulatory domain (SOK1RD). CCM3 could be immunoprecipitated with full-length SOK1 and SOK1RD, but not with SOK1ΔC (Fig. 5E). Thus, the interaction between CCM3 and SOK1 is mediated by the C-terminal domain of the latter.

Fig. 4.

CCM3 is important for Golgi reassembly after dispersion. (A) SaOS2 cells stably transduced with control or CCM3 shRNA were treated with BFA (10 μg/μl) for 90 minutes at 37°C. BFA was then washed out and cells were incubated at 37°C for 45 minutes, 90 minutes or 180 minutes to allow Golgi reassembly. Cells were fixed and stained for GM130 by immunofluorescence and analyzed by confocal microscopy. Scale bar: 10 μm. (B) Quantification of Golgi dispersion of SaOS2 cells with transiently transfected control or CCM3 siRNA, and in stable cell populations with control or CCM3 shRNA, when left untreated (control), and when treated with BFA and recovered for the specified periods of time. An average of 300 cells was counted per condition in each experiment. The average and standard deviation of three independent experiments are shown. P<0.01 for CCM3 versus control siRNA, and CCM3 versus control shRNA, in the control and at times 45, 90 and 180 minutes. (C) SaOS2 cells transduced with control shRNA (first lane) or CCM3 shRNA (second and third lanes) were transfected with empty vector or HA-CCM3 expression vector. Extracts were prepared and a western blot analysis was performed using anti-HA, anti-CCM3 or anti-tubulin (as a loading control) antibodies. (D) Stable SaOS2 cell populations with CCM3 shRNA were transfected with HA-CCM3 or empty vector (HA). Cells were then subjected to the same treatment and analysis as in B. *P<0.01. (E) Quantification of Golgi orientation in GFP-positive SaOS2 cells stably expressing control or CCM3 shRNA, and transfected with GFP and either HA empty vector or HA-CCM3. Confluent monolayers of cells were serum deprived overnight, wounded, and treated with 10% serum for 90 minutes (serum) or left untreated (SD). After staining with anti-GM130, cells were analyzed by immunofluorescence and scored for Golgi orientation. An average of 80 GFP-positive cells was counted per condition in each experiment. Discontinuous lines mark basal levels expected for a random orientation of 25%. The average and standard deviation of three independent experiments are shown. N.S., P>0.05. (F) Quantification of cell migration in GFP-positive SaOS2 cells stably expressing control or CCM3 shRNA, and transfected with GFP and either HA empty vector or HA-CCM3. Confluent monolayers of cells were subject to wound-healing assays. Migration in wound-healing assays was quantified by measuring the number of pixels in the denuded area, and expressed as the denuded area at time 0 hours minus the denuded area at 48 hours. Three wells were counted per condition in each experiment. The average and standard deviation of three independent experiments are shown. N.S., P>0.05.

The domain of SOK1 that binds to CCM3 (amino acids 333 to 426) is C terminal with respect to its binding site for GM130 (amino acids 270 to 302) (Nogueira et al., 2008; Preisinger et al., 2004) and does not overlap with it. This is compatible with SOK1 binding to GM130 and CCM3 simultaneously. To determine if such a trimeric complex can exist in cells, we transfected epitope-tagged versions of SOK1 and CCM3 in HEK293 cells (Fig. 5F). SOK1 was recovered in immunoprecipitates of endogenous GM130 with the same efficiency as when transfected alone or with CCM3. By contrast, whereas CCM3 could be detected in the GM130 immunoprecipitate, its recovery was greatly increased when SOK1 was co-transfected. Furthermore, both SOK1 and GM130 were readily detected in immunoprecipitates of endogenous CCM3 in SaOS2 cells (Fig. 5G). These results demonstrate that CCM3 forms a complex with GM130 and strongly suggest that SOK1 (and probably other GCKIII proteins) bridge this interaction.

Fig. 5.

CCM3 forms a complex with the GCKIII proteins and cis-Golgi GM130 protein. (A) HEK293 cells were transfected with empty or HA-CCM3 expression vectors with or without FLAG-SOK1. Western blots of FLAG-SOK1, HA-CCM3 and tubulin were performed with total cell lysates or after anti-FLAG or anti-HA immunoprecipitations (IP). (B) HEK293 cells were transfected with GFP-CCM3 together with empty vector, HA-SOK1, HA-Mst3 or HA-Mst4. Western blots for GFP, HA-GCKIII and tubulin were performed with total cell lysates or after anti-HA immunoprecipitations. (C) Extracts were prepared from SaOS2 cells, and western blots for CCM3, SOK1, Mst3, Mst4 or tubulin were performed with total cell lysates or after the specified immunoprecipitations. (D) Extracts were prepared from SaOS2 cells, and western blots for Mst3 and CCM3 were performed after immunoprecipitation with non-specific or anti-CCM3 antibody. (E) Western blots for FLAG, HA and tubulin were performed of total cell lysates or immunoprecipitations of HEK293 cells transfected with empty or HA-CCM3 expression vectors with or without full-length FLAG-SOK1 (FL), the C terminus of FLAG-SOK1 (RD) or the catalytic domain of FLAG-SOK1 (ΔC). A schematic representation of the SOK1 fragments used is shown. (F) HEK293 cells were transfected with empty vectors, HA-CCM3, FLAG-SOK1 or both. Western blots were performed for GM130, FLAG-SOK1 and HA-CCM3 in total cell lysates or after anti-GM130 immunoprecipitations. (G) Extracts were prepared from SaOS2 cells, and western blots for CCM3, SOK1, GM130 and tubulin were performed with total cell lysates or after nonspecific, anti-CCM3 or anti-SOK1 immunoprecipitations.

Effects of CCM3 depletion on GCKIII proteins

The CCM3-interacting proteins SOK1 and Mst4 are important for Golgi assembly and cell orientation. Thus, we asked whether CCM3 could modulate their activities. Depletion of CCM3 induced inhibition of SOK1 kinase activity, both in HeLa and in SaOS2 cells (Fig. 6A). This inhibition correlated with downregulation of the levels of SOK1 and the other two GCKIII proteins, Mst3 and Mst4. This was not a non-specific effect of RNAi, because it was reversed by the expression of CCM3 (Fig. 6B). We studied further the mechanism of this downregulation of GCKIII proteins and found that depletion of CCM3 did not induce changes in SOK1 mRNA (Fig. 6C). We then asked whether downregulation could be due to destabilization of the proteins. Thus, we assessed the decay of Mst3 after protein synthesis inhibition with cycloheximide (CHX) (as Mst3 is the only GCKIII protein we could consistently detect by western blot in shCCM3 cells). Depletion of CCM3 induced a marked shortening of the half-life of the Mst3 protein (13.9 hours in control cells versus 5.5 hours in CCM3-depleted cells; Fig. 6D). To gain further insight into the mechanism of destabilization of GCKIII proteins in the absence of CCM3, we treated cells with the proteasome inhibitor MG132. Mst3 accumulated significantly in cells depleted of CCM3 but not in control cells, confirming that Mst3 degradation in the absence of CCM3 is mediated through the proteasome (Fig. 6E). Consistently, when we assessed the amount of ubiquitylated SOK1 in CCM3-depleted cells, we found that it was at least as much as in cells with normal amounts of CCM3, despite the reduced amount of SOK1. This was the case both in untreated cells and in cells treated with the proteasome inhibitor (Fig. 6F). We concluded that degradation of GCKIII proteins is mediated at least in part by ubiquitylation and degradation through the proteasome, and that this is enhanced in the absence of CCM3.

Fig. 6.

Depletion of CCM3 induces enhanced ubiquitylation and proteasome-dependent degradation of GCKIII proteins. (A) The kinase activity of SOK1 was determined after immunoprecipitation with the SOK1 antibody. Quantification of a representative experiment is shown. (B) SaOS2 cells stably transduced with control or CCM3 shRNA constructs were transfected with empty plasmid or HA-CCM3 expression vector. Western blots for GCKIII family kinases, GM130, HA-CCM3, total CCM3 and tubulin were performed. (C) Total RNA was prepared from SaOS2 and HeLa stable cell populations transduced with control or CCM3 shRNA, and the levels of SOK1, CCM3 and GAPDH mRNAs were determined by RT-PCR. (D) SaOS2 cells stably transduced with control or CCM3 shRNA were treated with 20 μg/ml CHX. Extracts were prepared at the indicated times of treatment, and western blots for total Mst3 and tubulin were performed. (E) SaOS2 cells stably transduced with control or CCM3 shRNA were treated with 10 μM MG132 for the indicated times. Mst3 and tubulin were analyzed with western blotting. (F) SaOS2 cells stably transduced with control or CCM3 shRNA were transiently transfected with HA-tagged ubiquitin (HA-Ubi) and incubated in the presence or absence of 10 μM MG132 for 9 hours. Extracts were immunoprecipitated with non-specific or anti-SOK1 antibody, and ubiquitin conjugates were detected with anti-HA antibody. Control western blots for SOK1 and tubulin were performed with total cell lysates.

To assess whether the stabilization of the kinase component of the GM130-GCKIII complex by CCM3 plays a crucial role in Golgi assembly and cell migration, we expressed all possible combinations of GCKIII proteins in CCM3-depleted cells. All combinations tested resulted in an increase in Golgi assembly, with the expression of the three proteins inducing levels of Golgi assembly indistinguishable from that of control cells (Fig. 7A).

Fig. 7.

Golgi disassembly in the absence of CCM3 is the result of GCKIII downregulation and disruption of the CCM3-GCKIII complex. (A) SaOS2 cells stably transduced with CCM3 or control shRNA were transfected with empty vector and, for CCM3 shRNA, with HA-SOK1, HA-Mst3 and HA-Mst4 in the indicated combinations. After staining with anti-GM130, cells were analyzed by immunofluorescence and scored for Golgi dispersion. An average of 400 cells was counted per condition in each experiment. The average and standard deviation of three independent experiments are shown. N.S., P>0.05. (B) SaOS2 cells were transfected with empty vector or GFP-SOK1-RD plasmid. Western blots for GM130, SOK1, CCM3 and GFP were performed for whole cell lysates (upper panels); for SOK1 and CCM3 after immunoprecipitation with an anti-SOK1 antibody or an unrelated antibody (middle panels); and for SOK1, CCM3 and GFP after immunoprecipitation with an anti-CCM3 antibody or an unrelated antibody (lower panels). (C) SaOS2 cells stably transduced with CCM3 shRNA were transfected with GFP or GFP-SOK1-RD. Cells were either untreated (control) or incubated with BFA for 90 minutes, then washed and recovered for 180 minutes. After staining with anti-GM130, cells were analyzed by immunofluorescence and GFP-positive cells scored for Golgi dispersion. An average of 80 GFP-positive cells was counted per condition in each experiment. The average and standard deviation of three independent experiments are shown. *P<0.01.

Fig. 8.

Depletion of CCM3 induces Golgi dispersion through inhibition of 14-3-3ζ phosphorylation. (A) Extracts from SaOS2 and HeLa cells stably transduced with control or CCM3 shRNA were analysed by immunoblotting using anti-phosphoSer58-14-3-3ζ and anti-tubulin antibodies. As a positive control for serine 58 phosphorylation, SaOS2 control shRNA was treated with 500 μM H2O2 for 40 minutes. (B) SaOS2 stable cell populations with CCM3 shRNA were transiently transfected with 14-3-3ζ-WT (wild type), 14-3-3ζ-S58D, 14-3-3ζ-S58A or empty vector. Cells were fixed and stained for GM130 by immunofluorescence and analyzed by confocal microscopy. An average of 300 cells was counted per condition in each experiment. The average and standard deviation of three independent experiments are shown. *P<0.01.

The above results show that GCKIII proteins can rescue the Golgi phenotype induced by CCM3 depletion and suggest that CCM3 induces its effects through binding to this family of proteins on the cis face of the Golgi. We reasoned that, if this were the case, dissociation of CCM3 from GCKIII proteins should have the same effect as CCM3 depletion. Thus, we disrupted the GCKIII-CCM3 interaction by overexpressing SOK1RD, the C-terminal domain of SOK1 that binds CCM3. As expected, SOK1RD could bind to endogenous CCM3 (Fig. 7B, lower panels). Importantly, expression of SOK1RD clearly reduced the amount of CCM3 bound to endogenous SOK1 (Fig. 7B, middle and lower panels). Consistent with the binding of CCM3 to GCKIII proteins being important for its effects, levels of SOK1 were also greatly reduced (Fig. 7B, upper panels). Furthermore, the percentage of cells with disassembled Golgi was significantly higher in cells expressing SOK1RD than in control cells (Fig. 7C).

Having established that the effects of CCM3 on Golgi assembly are mediated through its binding and modulation of GCKIII proteins, we asked what event downstream of these kinases could be important for this effect. 14-3-3ζ has been shown to be phosphorylated at serine 58 by SOK1, both in untreated cells and after oxidative stress (Preisinger et al., 2004; Zhou et al., 2009). Consistent with 14-3-3ζ being a target of GCKIII proteins, its phosphorylation at serine 58 was diminished in CCM3-depleted cells (Fig. 8A). More significantly, transfection of the 14-3-3ζ phosphorylation-mimicking mutant S58D (but not wild-type 14-3-3ζ or the non-phosphorylatable S58A mutant) rescued the phenotype of CCM3 knockdown (Fig. 8B). Thus, the effects of CCM3 on Golgi assembly are mediated at least in part through 14-3-3ζ phosphorylation at serine 58.

Fig. 9.

Clinically relevant mutants of CCM3 are unable to bind to GCKIII proteins, upregulate GCKIII levels or rescue the Golgi phenotype of CCM3-depleted cells. (A) HA empty vector, HA-CCM3-WT, HA-CCM3-196 or HA-CCM3-203 vectors were transfected in SaOS2 cells stably transduced with CCM3 shRNA. Cell extracts were analyzed by immunoblotting using anti-HA, anti-CCM3 and anti-tubulin antibodies. A schematic representation of wild-type CCM3 (WT) and the mutants (196 and 303) used in this study is shown. (B) Cells from A were treated with BFA for 90 minutes and then recovered after 45 minutes, 90 minutes or 180 minutes. After staining for GM130, cells were scored for Golgi dispersion. An average of 300 cells was counted per condition in each experiment. The average and standard deviation of three independent experiments are shown. P<0.01 for HA-empty, HA-CCM3-196 and HA-CCM3-203 versus HA-CCM3-WT for the control and at times 45 minutes, 90 minutes and 180 minutes. (C) HEK293 cells were transfected with HA empty vector, HA-CCM3-WT, HA-CCM3-196 or HA-CCM3-203. Western blots were performed for HA, SOK1 and tubulin in total cell lysates or after anti-SOK1 immunoprecipitations.

Clinically relevant CCM3 mutants cannot rescue the CCM3 depletion phenotype

If the cellular phenotype of CCM3 depletion we have described is relevant to CCM pathogenesis, then clinically relevant CCM3 mutants should not be able to rescue it. Most CCM3 mutations are deletions of the whole gene or premature stop codons that result in highly defective truncated proteins. However, two different mutations in codons 196 and 203 have been described. Although both result in premature stop codons, the mutated genes encode proteins that are close in length to the wild-type protein (212 residues) (Fig. 9A) (Bergametti et al., 2005; Liquori et al., 2006). When we expressed these mutants, we found that their levels of expression were similar to that of wild-type CCM3 (Fig. 9A). Strikingly, the mutants were unable to rescue the defect in Golgi assembly induced by CCM3 depletion (Fig. 9B); this correlated with the inability of both mutants to bind to SOK1 efficiently (Fig. 9C) or to restore SOK1 expression to normal levels (Fig. 9A). The fact that mutants of CCM3 that lead to CCM formation were unable to rescue the Golgi phenotype induced by the depletion of wild-type CCM3 suggests that this phenotype is central to the pathophysiology of cavernous malformations. Finally, the inability of the CCM3 mutants to bind SOK1, to restore SOK1 levels to normal or to rescue the Golgi phenotype is consistent with the function of CCM3 in Golgi assembly being mediated by its binding to, and stabilization of, SOK1 and other GCKIII proteins.

Discussion

CCMs are vascular lesions in which endothelial cells form large caverns instead of normal capillaries. Because of the nature and location of the lesion, direct biochemical and cellular study of these abnormalities has been difficult. However, the realization that some CCMs have a genetic basis, and the identification of three CCM loci, has allowed the modeling of the disease at both the whole-animal and cellular level by inactivation of the relevant genes. Studies inactivating CCM1 and CCM2 have shown that these genes are important for cytoskeletal regulation. Accompanying evidence indicates that their products interact with endothelial transmembrane receptors, which suggests that endothelial cell shape and signaling might underlie at least some forms of the disease. However, no study showing a similar function for CCM3 has been reported so far.

In this paper, we show that CCM3 is important for Golgi assembly and for centrosome and Golgi orientation in wound-healing assays. Independently of its role in directed cell migration, the adequate positioning of centrosome and Golgi has been proposed to reflect the capability of the cell to polarize (Yadav et al., 2009); therefore, lack of polarizing ability is likely to be an important consequence of CCM3 inactivation. The two functions, cell orientation and Golgi assembly, are likely to be related, as the Golgi apparatus needs to disassemble and reassemble to change its position within the cell when the cell undergoes reorientation, and the integrity and position of the Golgi complex are known to be important for this orientation and directed migration (Vinogradova et al., 2009; Yadav et al., 2009). The effects of CCM3 on the Golgi apparatus are also likely to be linked to CCM pathogenesis, as two different clinically important mutants cannot rescue the effects of CCM3 knockdown on the Golgi.

Strong circumstantial evidence points to the Golgi apparatus as the crucial site of action of the GCKIII-CCM3 complex. The effects caused by CCM3 depletion are compatible with Golgi disassembly being the primary effect. In addition, GCKIII proteins and 14-3-3ζ phosphorylation are important for CCM3 functions, and these proteins have been localized to the Golgi (Preisinger et al., 2004; this article). Although there might be a pool of CCM3 that is not associated with the Golgi apparatus, it is clear that, independently of its location, CCM3 exerts the effects we report here through the binding and modulation of GCKIII proteins and the phosphorylation of 14-3-3ζ. Importantly, CCM3 does not overly affect the activity of the small G proteins Rho and cdc42, or the actin cytoskeleton, which suggests that it is not acting in concert with CCM1 and CCM2.

The relevant biochemical effect of CCM3 with respect to GCKIII proteins seems to be to stabilize them, because, in the absence of CCM3, both the levels and kinase activity of all three GCKIII proteins are downregulated due to destabilization of the proteins. Given that this facilitated degradation occurs through a proteasome- and ubiquitin-dependent pathway, CCM3 is likely to exert its function by protecting GCKIII proteins from ubiquitin ligation. Whether it directly impairs the action of an E3 ligase or inhibits a modification that marks GCKIII proteins for ubiquitylation will be the subject of future studies that will require the identification of the relevant E3 ligase.

The identification of the specific GCKIII protein or proteins that are important for CCM3 action will also require future research. We show here that only the expression of all three family members (SOK1, Mst3 and Mst4) fully restores normal Golgi assembly, but dissection of the contribution of each GCKIII protein to the phenotype induced by CCM3 depletion will require inhibition, not overexpression, of each individual kinase. However, SOK1 is the most likely GCKIII protein to be important in this respect. First, SOK1, but not the other two family members (Mst3 and Mst4), has been shown to be involved in Golgi assembly and cell orientation (Preisinger et al., 2004). Second, our results show that phosphorylation of 14-3-3ζ at serine 58 is an important downstream event for the CCM3 phenotype and SOK1 is the only GCKIII protein that has been shown to phosphorylate 14-3-3ζ.

Phosphorylation of 14-3-3ζ at serine 58 is an important consequence of GCKIII activity. Because of the pleiotropic nature of 14-3-3 proteins, it is difficult to pinpoint the mechanism through which this modification can be linked to Golgi assembly and cell orientation. However, it should be noted that 14-3-3ζ has been shown to bind to proteins important for cell polarity, such as par3 (Hurd et al., 2003). Intriguingly, besides its role in cell polarity, par3 is associated with tight junctions and is important for their assembly (Anderson and Van Itallie, 2008). Importantly, one of the characteristic features of the cells that form cerebral cavernomas is their aberrant tight junctions, which result in a leaky blood-brain barrier (Clatterbuck et al., 2001).

It is possible that a defect in cell polarity might also underlie the pathogenesis of other CCM syndromes. For instance, cells deficient in CCM2 have low activity of cdc42, a master regulator of cell polarity (Whitehead et al., 2009). Because depletion of CCM3 affects cell orientation without having a detectable effect on the activity of cdc42, different CCM proteins might be inducing the same or a very similar cellular effect through different mechanisms.

In summary, our findings demonstrate that clinically relevant mutations of CCM3 are unable to rescue the phenotypes that result from knockdown of CCM3. These abnormalities are likely to play a role in the pathogenesis of cerebral cavernomas and, if confirmed, this could set the stage for corrective therapeutic strategies aimed at CCM3.

Materials and Methods

Antibodies, plasmids and drugs

The antibodies used in this study are: rabbit polyclonal CCM3 (Proteintech Group); rabbit polyclonal GM130 CB-1008 (Calbiochem); mouse monoclonal FLAG M2, mouse monoclonal Golgi 58K clone 58K-9, mouse monoclonal α-tubulin clone B-5-1-2 and mouse monoclonal γ-tubulin clone GTU-88 (Sigma-Aldrich); goat polyclonal CCM3 (sc-67908), goat polyclonal SOK1 (sc-6865), goat polyclonal MST3 (sc-21400), goat polyclonal MST4 (sc-7150), goat polyclonal GM130 (sc-16268), mouse monoclonal anti-phospho-ERK (sc-7383) and mouse monoclonal HA probe (sc-7392) (SantaCruz Biotechnology); rabbit monoclonal ERK1-2 4695, rabbit polyclonal MST3 3723 and rabbit polyclonal MST4 3822 (Cell Signaling Technology); mouse monoclonal SOK1 clone 1G6 (Abnova); mouse monoclonal cytochrome c clone 7H8.2C12 (Becton Dickinson Biosciences); rabbit polyclonal GFP (ab290) (Abcam); and rabbit polyclonal 14-3-3ζ-phospho-Ser58 (PA1-4612) (Affinity BioReagents). The secondary antibodies used are: donkey anti-goat AlexaFluor488, goat anti-mouse AlexaFluor488 (Molecular Probes); goat anti-rabbit Cy3 (Jackson ImmunoResearch Laboratories); goat anti-rabbit horseradish peroxidase (HRP), goat anti-mouse HRP (Pierce).

All plasmids were constructed using standard molecular biology techniques. The oligonucleotides designed for the different mutants were purchased from Thermo Scientific (sequence available upon request).

BFA was from Sigma-Aldrich. Cells were treated with BFA (10 μg/μl for SaOS2 cells or 5 μg/μl for HeLa cells) for 90 minutes (SaOS2 cells) or 30 minutes (HeLa cells) at 37°C to disperse Golgi protein. BFA was washed out and cells were incubated at 37°C for 45 minutes, 90 minutes or 180 minutes in a complete culture medium to allow the Golgi reassembly process. Cells were treated with 20 μg/ml CHX from Sigma-Aldrich. Cells were treated with MG132 (10 μM) from Calbiochem.

siRNA- and shRNA-mediated knockdown

All chemically synthesized siRNA was purchased from Dharmacon (siRNA CCM3 ON-TARGETplus and siRNA control ON-TARGETplus) and transfected using a cell line nucleofector kit with Nucleofector II from Amaxa Biosystems. Stable cell populations with silenced CCM3 or control were obtained through selection after lentiviral transduction using MISSION lentiviral Non-Target shRNA Control Transduction Particles or MISSION lentiviral shRNA CCM3 Transduction Particles against the 3′-UTR of human CCM3 from Sigma-Aldrich (TRC libraries).

RT-PCR analysis

Cells were harvested and RNA extracted using TRIzol reagent (Invitrogen). Reverse transcriptase (RT)-PCR was performed by standard procedures using adequate PCR amplification primers (sequence available upon request). PCR products were separated using 1.0% agarose gel electrophoresis and visualized by ethidium bromide staining.

Cell culture and wound-healing assay for cell migration

HEK293 cells (European Collection of Cell Cultures), SaOS2 cells and HeLa cells (American Type Culture Collection) were grown as recommended.

For wound-healing assays, HeLa and SaOS2 cells were grown to confluence, and serum starved in 0.1% FBS overnight. Monolayer wounds were produced using a 200 μl pipette tip scratched through the center of the well. Photomicrographs were taken of the initial wound for comparison. Cells were then treated with either 0.1% FBS (SD) or 10% FBS (serum), and allowed to migrate into denuded areas for 24 hours in HeLa cells or 48 hours in SaOS2 cells. Cell migration was monitored using an inverted microscope (Olympus IX70) with a 4×/0.10 objective using a Hoffman modulation contrast system. Photoimages were acquired using an Olympus DP digital camera. The distance of migration was measured, using ImageJ software, as the number of pixels within the denuded area at time 0 hours minus the number of pixels within the denuded area at time 24 hours for HeLa and 48 hours for SaOS2 cells.

Immunofluorescence and image analysis

Cells were cultured on coverslips and fixed for 15 minutes in paraformaldehyde (PFA) 4%, permeabilized for 10 minutes in PBS with 0.25% Triton X-100, and incubated with the indicated antibodies followed by fluorescent secondary antibodies. DNA was stained with Hoechst33342. The coverslips were mounted in aqueous medium with anti-fading agents (Gel-Mount). Confocal images were collected using a Leica confocal microscope equipped with an HCX PL APO CS 63×/1.32 objective. LCS software was used for acquisition and analysis. Images are combinations of optical sections taken in the z-axis at 0.5 μm intervals. For all microscope photographs, Adobe Photoshop software was used to cut, resize and mount the photographs in the figures.

Fluorescent staining of actin

For actin staining, cells grown on glass slides were fixed for 15 minutes in PFA 2%, permeabilized for 10 minutes in PBS with 0.5% Triton X-100 and then incubated for 45 minutes with 10 μg/μl of phalloidin-TRITC (Sigma). The cells were then mounted in aqueous medium with anti-fading agents (Gel-Mount) and examined by fluorescent microscopy.

Quantification of Golgi and centrosome polarization, and Golgi dispersion

The Golgi was stained with GM130 antibody and centrosomes with γ-tubulin antibody. Golgi and centrosome orientation was determined for the first row of cells facing the wound, as described previously (Etienne-Manneville and Hall, 2001), and was counted as oriented if more than half was located in a 90° sector emerging from the center of the nucleus and facing the wound edge. Basal levels of expected random orientation of 25% are marked by discontinuous lines in the graphs. The percentage of Golgi or centrosome orientation was calculated by dividing the number of oriented cells by the number of total cells for each condition. A minimum of 100 cells was counted for each condition and experiment, unless otherwise specified. The mean percentage from four independent experiments was averaged, and presented as a mean and standard deviation.

Golgi was considered dispersed when the GM130 staining or Golgi 58K staining spanned more than 5 μm at its longest diameter. A minimum of 300 cells was counted for each condition and experiment, unless otherwise specified. The mean percentage from four independent experiments was averaged, and presented as a mean and standard deviation.

Transfections, immunoprecipitation and immunoblotting

HEK293 cells were transfected using the calcium phosphate protocol. SaOS2 cells were transfected using the cell line nucleofector kit with Nucleofector II from Amaxa Biosystems following manufacturer's instructions.

Western blotting and immunoprecipitations were performed by standard procedures, after preparation of extracts in lysis buffer [20 mM HEPES pH 7.4, 2 mM EGTA, 50 mM β-glycerophosphate, 1 mM sodium orthovanadate, 1% Triton X-100, 10% glycerol, 1 mM dithiothreitol (DTT), 400 μM PMSF, 2 μM pepstatin, 2.3 μg/ml aprotinin, 2 μM leupeptin].

Kinase assays

Extracts were immunoprecipitated with anti-SOK1 antibody and kinase assays were performed as described (Pombo et al., 1996). Briefly, immune complexes were collected with protein G-sepharose beads. Beads were washed three times in lysis buffer, three times in LiCl buffer (500 mM LiCl, 2 mM DTT, 100 mM Tris-HCl pH 7.6) and three times in assay buffer (20 mM MOPS pH 7.2, 2 mM EGTA, 10 mM MgCl2, 0.1% Triton X-100, 1 mM DTT). Kinase assays were started by the addition of myelin basic protein (MBP) and [γ-32P]ATP (100 μM, 3000-9000 c.p.m./pmol). After 5 minutes at 30°C, the kinase reactions were stopped with Laemmli sample buffer. Following SDS-PAGE and autoradiography, the bands corresponding to the substrate were cut out of the gel and radioactivity was determined by liquid scintillation counting in a 1414 liquid scintillation counter (Wallac-Winspectral).

Isolation of Golgi membranes

Liver from Sprague-Dawley rats was used to obtain enriched Golgi fractions using discontinuous sucrose gradient centrifugation (Bonifacino et al., 2004). Briefly, 10 g of liver was minced and suspended in 40 ml of homogenization medium (HM: 0.25 mM sucrose, 10 mM Tris-Cl pH 7.4). The sample was homogenized using a Potter-Elvehjem homogenizer with five up-and-down strokes of the pestle, rotating at 500 rpm. The homogenate was centrifuged at low speed (1000 g for 10 minutes at 4°C). The nuclear pellet was transferred in 20 ml of HM to a Dounce homogenizer and then centrifuged at low speed. Previous and last supernatants were combined and centrifuged (3000 g for 10 minutes at 4°C). The resulting supernatant was centrifuged in a high-speed centrifuge (17,000 g for 20 minutes at 4°C). The pellet was resuspended in 2 ml of HM, with 8 ml sucrose (2.0 M) added, and homogenized with a Dounce homogenizer. 5 ml of homogenate was loaded in a discontinuous sucrose gradient (4 ml 1.33 M, 2 ml 1.2 M, 2 ml 1.1 M, 2 ml 0.77 M and 0.25 M to fill the tube) and ultracentrifuged (100,000 g for 1 hour at 4°C). The enriched Golgi membrane fractions were collected at the 0.77 M-1.1 M and 1.1 M-1.2 M interfaces. Analysis was carried out by SDS-PAGE and immunoblotting.

Determination of RhoA and Cdc42 activity

Activation assays for the small GTPases were performed using the G-LISA RhoA or Cdc42 luminescence-based Biochem kits (Cytoskeleton). Briefly, SaOS2 cells stably transduced with control or CCM3 shRNA were grown in a six-well cluster plate, serum starved for 24 hours, then treated with 10% serum for 3 minutes (serum) or left untreated, lysed in the provided lysis buffer (including protease inhibitors) and centrifuged to obtain the soluble extract. After correction of the total protein concentration according to the manufacturer's instructions, 0.25 mg/ml of each sample was added to the ELISA plates. Wells were washed and active GTPase was detected by specific antibodies according to the manufacturer's instructions in a luminescent reaction and measured in a Sunrise microplate reader (Tecan).

Statistical analysis

The statistical significance of all data obtained was assessed by Mann-Whitney tests. All data were analyzed using SPSS software version 12.0.0.

Acknowledgements

We thank Anxo Vidal and Gonzalo Egea for helpful discussions, and Carme Caelles (Instituto de Recerca Biomédica, Barcelona, Spain) for providing the pCDNA3-HA-ubiquitin construct. This work was supported by grants from the Instituto de Salud Carlos III of Spain (PI080655, co-financed with Regional Development European Funds) and from the Xunta de Galicia (PGIDT06PXIB208061PR to J.Z. and INCITE09 208 110 PR to C.P.). M.F. is a predoctoral fellow from the Ministerio de Educación y Ciencia of Spain.

Footnotes

  • Accepted January 14, 2010.

References

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