The cerebral cavernous malformation proteins CCM2L and CCM2 prevent the activation of the MAP kinase MEKK3

Abstract

Three genes, CCM1, CCM2, and CCM3, interact genetically and biochemically and are mutated in cerebral cavernous malformations (CCM). A recently described member of this CCM family of proteins, CCM2-like (CCM2L), has high homology to CCM2. Here we show that its relative expression in different tissues differs from that of CCM2 and, unlike CCM2, the expression of CCM2L in endothelial cells is regulated by density, flow, and statins. In vitro, both CCM2L and CCM2 bind MEKK3 in a complex with CCM1. Both CCM2L and CCM2 interfere with MEKK3 activation and its ability to phosphorylate MEK5, a downstream target. The in vivo relevance of this regulation was investigated in zebrafish. A knockdown of ccm2l and ccm2 in zebrafish leads to a more severe "big heart" and circulation defects compared with loss of function of ccm2 alone, and also leads to substantial body axis abnormalities. Silencing of mekk3 rescues the big heart and body axis phenotype, suggesting cross-talk between the CCM proteins and MEKK3 in vivo. In endothelial cells, CCM2 deletion leads to activation of ERK5 and a transcriptional program that are downstream of MEKK3. These findings suggest that CCM2L and CCM2 cooperate to regulate the activity of MEKK3.

Keywords: MAP kinase; cerebral cavernous malformation; endothelium; expression; signaling.

Fig. 1.

CCM2L expression and interaction with CCM1. (A) Exons with identical amino acid sequences for canonical isoform 1 (62 kD) and shorter isoform 2 (46 kD) are in blue. Sequences only in isoform 2 are in black. The PTB domain is underlined, and the gray boxes represent the 3′ UTR. (B) CCM2L and CCM2 mRNA analysis by qRT-PCR using primers for CCM2 and both CCM2L isoforms in endothelial cells (HUVEC), cord blood endothelial progenitor cells (cbEPC), peripheral blood mononuclear cells (PBMCs), neutrophils (PMN), or indicated samples of human tissue. (C) HUVEC were plated at confluency (1:1) or at indicated dilutions for 24 or 48 h. mRNA levels were evaluated by qRT-PCR. (D) HUVEC were treated with vehicle control, simvastatin, or atorvastatin at indicated µM concentrations, or subjected to shear stress (LSS) for 24 h, followed by analysis of CCM2L and CCM2 mRNA levels by qRT-PCR. (E) HEK293 cells were transfected with Myc-tagged CCM1, Flag-tagged CCM2L, or Flag-tagged CCM2 as indicated. Cell lysates were subjected to IP with anti-tag antibodies, followed by Western blot analysis (WB) with indicated antibodies. CCM1 coimmunoprecipitated with CCM2L and CCM2. n = 3 independent experiments.

Fig. 2.

CCM2L and CCM2 interact with MEKK3 and prevent its autophosphorylation. HEK293 cells were transfected with the indicated Myc-, Flag (Flg)-, or T7-tagged constructs alone or in combination. Anti-tag immunoprecipitated proteins and cell lysates were analyzed by Western blot (WB). (A) CCM2L and CCM2 interact with MEKK3 (K3). (B) CCM2L and CCM2 interact with only the N-terminal domain of MEKK3 (K3ΔK, lacking the kinase domain), and not with the kinase domain (K3CA, catalytically active kinase domain). (C) CCM2L isoform 1 (-1) and isoform 2 (-2) interact with CCM1, but only isoform 1 (-1) binds MEKK3. (D) CCM2L, but not CCM2L4xM (with mutations in four hydrophobic residues), interacts with MEKK3. (E) MEKK3 is in a complex with CCM1 only when CCM2L or CCM2 are present. (F) CCM2L (Upper) and CCM2 (Lower) prevent MEKK3 autophosphorylation. Immunoprecipitated MEKK3 and kinase dead MEKK3 (K3K > A) alone or with CCM2L or CCM2 were subjected to an in vitro kinase assay (+ATP). MEKK3 p-Ser526 status was analyzed by Western blot analysis. MEKK3, but not MEKK3K > A, shows a slower migrating band, indicating autophosphorylated MEKK3 (bar). MEKK3 present in CCM2L or CCM2 immunoprecipitates is unable to autophosphorylate (arrow), and MEKK3 complexed with CCM2L has less p-Ser526. (G) MEKK3 (K3), MEK5 (K5), and CCM2L are present in a complex. Anti-CCM2L immunoprecipitates contain MEK5 only when MEKK3 is also cotransfected. (H) MEK5 phosphorylation by MEKK3 is reduced in the presence of CCM2L. In vitro kinase assays of indicated immunoprecipitates were followed by WB using anti-tag antibodies and p-MEK5–specific antibody (Ser311/Thr315). (I) Cells were transfected with MEKK3 with and without CCM2L at the indicated plasmid ratios. Aliquots of cell lysates containing similar amounts of MEKK3 (Lower) were evaluated for MEKK3 p-Ser526 (Upper). Densitometric analyses of WBs are reported as fold reduction in CCM2L-containing samples. n = 3–4 independent experiments for each panel.

Fig. S1.

(A) HUVEC were transduced with adenoviral control or adenovirus expressing FLAG (Flg)-tagged CCM2L and harvested 48 h later. Western blot analysis of anti-Flag immunoprecipitates show that endogenous MEKK3 interacts with CCM2L. (B) CCM2L was transiently transfected together with MEKK3 (K3) or MEKK3 kinase dead (K3 K > A). MEKK3 kinase dead is more highly represented than MEKK3 in the CCM2L immunoprecipitates. CCM2L phosphorylation specifically by active MEKK3 results in reduced mobility in SDS gels (bar).

Fig. 3.

Genetic interaction of ccm2l with ccm1 and ccm2 and modulation of mekk3 activity in zebrafish. (A) Single-cell flk-GFP embryos were injected with control (ctrl) or ccm2l MO (MO-1), and images were obtained at 48 h postfertilization. The arrow indicates cardiac enlargement/big heart. (B) The big heart phenotype was evaluated with increasing doses of ccm2l MO-1. (C–E) Different amounts of ccm2l MO-1 or ctrl MO were injected into embryos from san/ccm1 (C) and vtn/ccm2 (D) heterozygous crosses. The vtn/ccm2 zebrafish with ccm2l knockdown show further enlargement of the heart in combination with heart failure (HF) and tail/axis defects (arrows). Phenotypes were classified as WT (no phenotype), big heart, HF/axis (HF and axis/tail defects), or dead (lack of survival). (E) Representative pictures of vtn/ccm2 mutants without (Left) or with ccm2l MO-1 injected (Right). (F) Rescue of the axis phenotype in ccm2/ccm2l-deficient fish with 4 ng/µL mekk3 MO. Embryos from vtn/ccm2 heterozygous crosses were injected with ccm2l alone or in combination with ctrl or mekk3 MO, and the axis defect phenotype was scored. (G) Rescue of the big heart phenotype in vtn/ccm2 mutant fish (vtn) with ctrl or 2 ng/µL mekk3 MO. (H) Rescue of big heart in WT plus 4 ng/µL ccm2l MO-1 injection with ctrl MO or indicated concentrations of mekk3 MO. (I and J) Rescue of big heart (I) and tail axis defects (J) in vtn + ccm2l MO-1 zebrafish, with human CCM2L mRNA. The big heart phenotype in vtn alone (I, −) is shown for comparison. *P < 0.05, unpaired t test. Graphs represent data pooled from three independent experiments. Error bars represent SEM.

Fig. S2.

(A) Dose–response curve of ccm2l MO-2 injected into embryos from WT. ***P < 0.005, ANOVA. (B) Representative pictures of WT embryos (Upper) with and without ccm2l MO-2 (Lower). The arrow indicates big heart. (C and D) Injection of MO-2 in vtn/ccm2 heterozygous crosses results in heart failure and severe tail/axis defects. Quantitation of phenotypes in C: WT, no phenotype; big heart; HF/axis, heart failure and axis/tail defects; dead, lack of survival. Representative images are shown in D. (E) Genotyping. Chromatograms representing sequence results from vtn heterozygous crosses injected with ccm2l MO-1 (Upper). vtn heterozygous embryos show a double peak (C and A, asterisks), inferring the presence of a vtn (A) and an ortholog WT (C) gene, whereas WT controls show a single peak (C). vtn homozygous embryos were not detected in the surviving embryos in this cross, which may reflect lower survival rates in this group after ccm2l MO injection (Fig. 2D). (F and G) Injections of a cardiac-specific MO (troponinT, tnnt2) in vtn mutants (F) and ccm2l MO-1 injections in silent heart mutant (G) did not phenocopy the ccm2l MO-1 axis defect results (ns, not significant, unpaired t test). Graphs represent data pooled from three independent experiments. The number of embryos scored is in parentheses. Error bars represent SEM. (H) Representative picture of mekk3 mRNA-injected vtn/ccm2 embryos showing more severe tail axis defects and cardiac failure.

Fig. 4.

ERK5 activation in CCM2 deleted endothelial cells. (A and B) HUVEC transduced with control or CCM2 exons 2 or 3 CRISPR/Cas9 (Crpr) lentivirus were analyzed for ERK activation by Western blot (A) or for KLF2 or KLF4 mRNA by qRT-PCR (B). Activation of ERK5 is detected by the appearance of a slower migrating form indicating phosphorylation (arrow). (C) HUVEC were transduced with lentiviral particles to express Myc-tagged MEKK3 and harvested for qRT-PCR after 48 h, and KLF2 and KLF4 mRNA levels were evaluated. ***P < 0.05. n = 4 experiments. Error bars represent SEM.