STK25 Protein Mediates TrkA and CCM2 Protein-dependent Death in Pediatric Tumor Cells of Neural Origin

The TrkA receptor tyrosine kinase induces death in medulloblastoma cells via an interaction with the cerebral cavernous malformation 2 (CCM2) protein. We used affinity proteomics to identify the germinal center kinase class III (GCKIII) kinases STK24 and STK25 as novel CCM2 interactors. Down-modulation of STK25, but not STK24, rescued medulloblastoma cells from NGF-induced TrkA-dependent cell death, suggesting that STK25 is part of the death-signaling pathway initiated by TrkA and CCM2. CCM2 can be phosphorylated by STK25, and the kinase activity of STK25 is required for death signaling. Finally, STK25 expression in tumors is correlated with positive prognosis in neuroblastoma patients. These findings delineate a death-signaling pathway downstream of neurotrophic receptor tyrosine kinases that may provide targets for therapeutic intervention in pediatric tumors of neural origin.     

Ccm3 functions in a manner distinct from Ccm1 and Ccm2 in a zebrafish model of CCM vascular disease

Cerebral cavernous malformations (CCMs) are vascular anomalies of the central nervous system that arise due to mutations in genes encoding three unrelated proteins: CCM1 (KRIT1); CCM2 (Malcavernin/OSM) and CCM3 (PDCD10). Both biochemical and mutant studies suggest that CCM1 and CCM2 act as part of a physical complex to regulate vascular morphogenesis and integrity. In contrast, mouse Ccm3 mutant and in vitro cell culture data suggests an independent role for Ccm3. In this study, we sought to use the zebrafish model system to examine for the first time the role of ccm3 in cranial vessel development. We report that inhibition of zebrafish ccm3a/b causes heart and circulation defects distinct from those seen in ccm1 (santa) and ccm2 (valentine) mutants, and leads to a striking dilation and mispatterning of cranial vessels reminiscent of the human disease pathology. ccm3, but not ccm2, defects can be rescued upon overexpression of stk25b, a GCKIII kinase previously shown to interact with CCM3. Morpholino knockdown of the GCKIII gene stk25b results in heart and vasculature defects similar to those seen in ccm3 morphants. Finally, additional loss of ccm3 in ccm2 mutants leads to a synergistic increase in cranial vessel dilation. These results support a model in which CCM3 plays a role distinct from CCM1/2 in CCM pathogenesis, and acts via GCKIII activity to regulate cranial vasculature integrity and development. CCM3/GCKIII activity provides a novel therapeutic target for CCMs, as well as for the modulation of vascular permeability.     

Structure-function analysis of core STRIPAK Proteins: a signaling complex implicated in Golgi polarization

Cerebral cavernous malformations (CCMs) are alterations in brain capillary architecture that can result in neurological deficits, seizures, or stroke. We recently demonstrated that CCM3, a protein mutated in familial CCMs, resides predominantly within the STRIPAK complex (striatin interacting phosphatase and kinase). Along with CCM3, STRIPAK contains the Ser/Thr phosphatase PP2A. The PP2A holoenzyme consists of a core catalytic subunit along with variable scaffolding and regulatory subunits. Within STRIPAK, striatin family members act as PP2A regulatory subunits. STRIPAK also contains all three members of a subfamily of Sterile 20 kinases called the GCKIII proteins (MST4, STK24, and STK25). Here, we report that striatins and CCM3 bridge the phosphatase and kinase components of STRIPAK and map the interacting regions on each protein. We show that striatins and CCM3 regulate the Golgi localization of MST4 in an opposite manner. Consistent with a previously described function for MST4 and CCM3 in Golgi positioning, depletion of CCM3 or striatins affects Golgi polarization, also in an opposite manner. We propose that STRIPAK regulates the balance between MST4 localization at the Golgi and in the cytosol to control Golgi positioning.     

Striatins Contain a Noncanonical Coiled Coil That Binds Protein Phosphatase 2A A Subunit to Form a 2:2 Heterotetrameric Core of Striatin-interacting Phosphatase and Kinase (STRIPAK) Complex

The protein phosphatase 2A (PP2A) and kinases such as germinal center kinase III (GCKIII) can interact with striatins to form a supramolecular complex called striatin-interacting phosphatase and kinase (STRIPAK) complex. Despite the fact that the STRIPAK complex regulates multiple cellular events, it remains only partially understood how this complex itself is assembled and regulated for differential biological functions. Our recent work revealed the activation mechanism of GCKIIIs by MO25, as well as how GCKIIIs heterodimerize with CCM3, a molecular bridge between GCKIII and striatins. Here we dissect the structural features of the coiled coil domain of striatin 3, a novel type of PP2A regulatory subunit that functions as a scaffold for the assembly of the STRIPAK complex. We have determined the crystal structure of a selenomethionine-labeled striatin 3 coiled coil domain, which shows it to assume a parallel dimeric but asymmetric conformation containing a large bend. This result combined with a number of biophysical analyses provide evidence that the coiled coil domain of striatin 3 and the PP2A A subunit form a stable core complex with a 2:2 stoichiometry. Structure-based mutational studies reveal that homodimerization of striatin 3 is essential for its interaction with PP2A and therefore assembly of the STRIPAK complex. Wild-type striatin 3 but not the mutants defective in PP2A binding strongly suppresses apoptosis of Jurkat cells induced by the GCKIII kinase MST3, most likely through a mechanism in which striatin recruits PP2A to negatively regulate the activation of MST3. Collectively, our work provides structural insights into the organization of the STRIPAK complex and will facilitate further functional studies.     

Adaptor protein cerebral cavernous malformation 3 (CCM3) mediates phosphorylation of the cytoskeletal proteins ezrin/radixin/moesin by mammalian Ste20-4 to protect cells from oxidative stress

While studying the functions of CCM3/PDCD10, a gene encoding an adaptor protein whose mutation results in vascular malformations, we have found that it is involved in a novel response to oxidative stress that results in phosphorylation and activation of the ezrin/radixin/moesin (ERM) family of proteins. This phosphorylation protects cells from accidental cell death induced by oxidative stress. We also present evidence that ERM phosphorylation is performed by the GCKIII kinase Mst4, which is activated and relocated to the cell periphery after oxidative stress. The cellular levels of Mst4 and its activation after oxidative stress depend on the presence of CCM3, as absence of the latter impairs the phosphorylation of ERM proteins and enhances death of cells exposed to reactive oxygen species. These findings shed new light on the response of cells to oxidative stress and identify an important pathophysiological situation in which ERM proteins and their phosphorylation play a significant role.     

Crystal Structure of CCM3, a Cerebral Cavernous Malformation Protein Critical for Vascular Integrity

CCM3 mutations are associated with cerebral cavernous malformation (CCM), a disease affecting 0.1–0.5% of the human population. CCM3 (PDCD10, TFAR15) is thought to form a CCM complex with CCM1 and CCM2; however, the molecular basis for these interactions is not known. We have determined the 2.5 Å crystal structure of CCM3. This structure shows an all α-helical protein containing two domains, an N-terminal dimerization domain with a fold not previously observed, and a C-terminal focal adhesion targeting (FAT)-homology domain. We show that CCM3 binds CCM2 via this FAT-homology domain and that mutation of a highly conserved FAK-like hydrophobic pocket (HP1) abrogates CCM3-CCM2 interaction. This CCM3 FAT-homology domain also interacts with paxillin LD motifs using the same surface, and partial CCM3 co-localization with paxillin in cells is lost on HP1 mutation. Disease-related CCM3 truncations affect the FAT-homology domain suggesting a role for the FAT-homology domain in the etiology of CCM.     

Molecular recognition of leucine-aspartate repeat (LD) motifs by the focal adhesion targeting homology domain of cerebral cavernous malformation 3 (CCM3)

Cerebral cavernous malformation (CCM) is a disease that affects between 0.1 and 0.5% of the human population, with mutations in CCM3 accounting for ∼15% of the autosomal dominant form of the disease. We recently reported that CCM3 contains an N-terminal dimerization domain (CCM3D) and a C-terminal focal adhesion targeting (FAT) homology domain. Intermolecular protein-protein interactions of CCM3 are mediated by a highly conserved surface on the FAT homology domain and are affected by CCM3 truncations in the human disease. Here we report the crystal structures of CCM3 in complex with three different leucine-aspartate repeat (LD) motifs (LD1, LD2, and LD4) from the scaffolding protein paxillin, at 2.8, 2.7, and 2.5 Å resolution. We show that CCM3 binds LD motifs using the highly conserved hydrophobic patch 1 (HP1) and that this binding is similar to the binding of focal adhesion kinase and Pyk2 FAT domains to paxillin LD motifs. We further show by surface plasmon resonance that CCM3 binds paxillin LD motifs with affinities in the micromolar range, similar to FAK family FAT domains. Finally, we show that endogenous CCM3 and paxillin co-localize in mouse cerebral pericytes. These studies provide a molecular-level framework to investigate the protein-protein interactions of CCM3.     

Deletions in CCM2 are a common cause of cerebral cavernous malformations

Cerebral cavernous malformations (CCMs) are vascular abnormalities of the brain that can result in a variety of neurological disabilities, including hemorrhagic stroke and seizures. Mutations in the gene KRIT1 are responsible for CCM1, mutations in the gene MGC4607 are responsible for CCM2, and mutations in the gene PDCD10 are responsible for CCM3. DNA sequence analysis of the known CCM genes in a cohort of 63 CCM-affected families showed that a high proportion (40%) of these lacked any identifiable mutation. We used multiplex ligation-dependent probe analysis to screen 25 CCM1, -2, and -3 mutation-negative probands for potential deletions or duplications within all three CCM genes. We identified a total of 15 deletions: 1 in the CCM1 gene, 0 in the CCM3 gene, and 14 in the CCM2 gene. In our cohort, mutation screening that included sequence and deletion analyses gave disease-gene frequencies of 40% for CCM1, 38% for CCM2, 6% for CCM3, and 16% with no mutation detected. These data indicate that the prevalence of CCM2 is much higher than previously predicted, nearly equal to CCM1, and that large genomic deletions in the CCM2 gene represent a major component of this disease. A common 77.6-kb deletion spanning CCM2 exons 2-10 was identified, which is present in 13% of our entire CCM cohort. Eight probands exhibit an apparently identical recombination event in the CCM2 gene, involving an AluSx in intron 1 and an AluSg distal to exon 10. Haplotype analysis revealed that this CCM2 deletion occurred independently at least twice in our families. We hypothesize that these deletions occur in a hypermutable region because of surrounding repetitive sequence elements that may catalyze the formation of intragenic deletions.     

Biallelic CCM3 mutations cause a clonogenic survival advantage and endothelial cell stiffening

CCM3, originally described as PDCD10, regulates blood‐brain barrier integrity and vascular maturation in vivo. CCM3 loss‐of‐function variants predispose to cerebral cavernous malformations (CCM). Using CRISPR/Cas9 genome editing, we here present a model which mimics complete CCM3 inactivation in cavernous endothelial cells (ECs) of heterozygous mutation carriers. Notably, we established a viral‐ and plasmid‐free crRNA:tracrRNA:Cas9 ribonucleoprotein approach to introduce homozygous or compound heterozygous loss‐of‐function CCM3 variants into human ECs and studied the molecular and functional effects of long‐term CCM3 inactivation. Induction of apoptosis, sprouting, migration, network and spheroid formation were significantly impaired upon prolonged CCM3 deficiency. Real‐time deformability cytometry demonstrated that loss of CCM3 induces profound changes in cell morphology and mechanics: CCM3‐deficient ECs have an increased cell area and elastic modulus. Small RNA profiling disclosed that CCM3 modulates the expression of miRNAs that are associated with endothelial ageing. In conclusion, the use of CRISPR/Cas9 genome editing provides new insight into the consequences of long‐term CCM3 inactivation in human ECs and supports the hypothesis that clonal expansion of CCM3‐deficient dysfunctional ECs contributes to CCM formation.     

A PP2A phosphatase high density interaction network identifies a novel striatin-interacting phosphatase and kinase complex linked to the cerebral cavernous malformation 3 (CCM3) protein

The serine/threonine protein phosphatases are targeted to specific subcellular locations and substrates in part via interactions with a wide variety of regulatory proteins. Understanding these interactions is thus critical to understanding phosphatase function. Using an iterative affinity purification/mass spectrometry approach, we generated a high density interaction map surrounding the protein phosphatase 2A catalytic subunit. This approach recapitulated the assembly of the PP2A catalytic subunit into many different trimeric complexes but also revealed several new protein-protein interactions. Here we define a novel large multiprotein assembly, referred to as the striatin-interacting phosphatase and kinase (STRIPAK) complex. STRIPAK contains the PP2A catalytic (PP2Ac) and scaffolding (PP2A A) subunits, the striatins (PP2A regulatory B' subunits), the striatin-associated protein Mob3, the novel proteins STRIP1 and STRIP2 (formerly FAM40A and FAM40B), the cerebral cavernous malformation 3 (CCM3) protein, and members of the germinal center kinase III family of Ste20 kinases. Although the function of the CCM3 protein is unknown, the CCM3 gene is mutated in familial cerebral cavernous malformations, a condition associated with seizures and strokes. Our proteomics survey indicates that a large portion of the CCM3 protein resides within the STRIPAK complex, opening the way for further studies of CCM3 biology. The STRIPAK assembly establishes mutually exclusive interactions with either the CTTNBP2 proteins (which interact with the cytoskeletal protein cortactin) or a second subcomplex consisting of the sarcolemmal membrane-associated protein (SLMAP) and the related coiled-coil proteins suppressor of IKKepsilon (SIKE) and FGFR1OP2. We have thus identified several novel PP2A-containing protein complexes, including a large assembly linking kinases and phosphatases to a gene mutated in human disease.     

Kinases in cerebral cavernous malformations: Pathogenesis and therapeutic targets

Cerebral cavernous malformations (CCMs) are low-flow, hemorrhagic vascular lesions of the central nervous system of genetic origin, which can cause stroke-like symptoms and seizures. From the identification of CCM1, CCM2 and CCM3 as genes related to disease progression, molecular and cellular mechanisms for CCM pathogenesis have been established and the search for potential drugs to target CCM has begun. Broadly speaking, kinases are the major group signaling in CCM pathogenesis. These include the MEKK3/MEK5/ERK5 cascade, Rho/Rock signaling, CCM3/GCKIII signaling, PI3K/mTOR signaling, and others. Since the discovery of Rho/Rock in CCM pathogenesis, inhibitors for Rho signaling and subsequently other components in CCM signaling were discovered and applied in preclinical and clinical trials to ameliorate CCM progression. This review discusses the general aspects of CCM disease, kinase-mediated signaling in CCM pathogenesis and the current state of potential treatment options for CCM. It is suggested that kinase target drug development in the context of CCM might facilitate and meet the unmet requirement – a non-surgical option for CCM disease.     

Structural Basis for the Disruption of the Cerebral Cavernous Malformations 2 (CCM2) Interaction with Krev Interaction Trapped 1 (KRIT1) by Disease-associated Mutations

Familial cerebral cavernous malformations (CCMs) are predominantly neurovascular lesions and are associated with mutations within the KRIT1, CCM2, and PDCD10 genes. The protein products of KRIT1 and CCM2 (Krev interaction trapped 1 (KRIT1) and cerebral cavernous malformations 2 (CCM2), respectively) directly interact with each other. Disease-associated mutations in KRIT1 and CCM2 mostly result in loss of their protein products, although rare missense point mutations can also occur. From gene sequencing of patients known or suspected to have one or more CCMs, we discover a series of missense point mutations in KRIT1 and CCM2 that result in missense mutations in the CCM2 and KRIT1 proteins. To place these mutations in the context of the molecular level interactions of CCM2 and KRIT1, we map the interaction of KRIT1 and CCM2 and find that the CCM2 phosphotyrosine binding (PTB) domain displays a preference toward the third of the three KRIT1 NPX(Y/F) motifs. We determine the 2.75 Å co-crystal structure of the CCM2 PTB domain with a peptide corresponding to KRIT1^NPX(Y/F)3, revealing a Dab-like PTB fold for CCM2 and its interaction with KRIT1^NPX(Y/F)3. We find that several disease-associated missense mutations in CCM2 have the potential to interrupt the KRIT1-CCM2 interaction by destabilizing the CCM2 PTB domain and that a KRIT1 mutation also disrupts this interaction. We therefore provide new insights into the architecture of CCM2 and how the CCM complex is disrupted in CCM disease.     

EphB4 forward signalling mediates angiogenesis caused by CCM3/PDCD10‐ablation

CCM3, also named as PDCD10, is a ubiquitous protein expressed in nearly all tissues and in various types of cells. It is essential for vascular development and post‐natal vessel maturation. Loss‐of‐function mutation of CCM3 predisposes for the familial form of cerebral cavernous malformation (CCM). We have previously shown that knock‐down of CCM3 stimulated endothelial angiogenesis via impairing DLL4‐Notch signalling; moreover, loss of endothelial CCM3 stimulated tumour angiogenesis and promoted tumour growth. The present study was designed to further elucidate the inside signalling pathway involved in CCM3‐ablation‐mediated angiogenesis. Here we report for the first time that silencing endothelial CCM3 led to a significant up‐regulation of EphB4 mRNA and protein expression and to an increased kinase activity of EphB4, concomitantly accompanied by an activation of Erk1/2, which was reversed by treatment with the specific EphB4 kinase inhibitor NVP‐BHG712 (NVP), indicating that silencing CCM3 activates EphB4 kinase forward signalling. Furthermore, treatment with NVP rescued the hyper‐angiogenic phenotype induced by knock‐down of endothelial CCM3 in vitro and in vivo. Additional study demonstrated that the activation of EphB4 forward signalling in endothelial cells under basal condition and after CCM3‐silence was modulated by DLL4/Notch signalling, relying EphB4 at downstream of DLL4/Notch signalling. We conclude that angiogenesis induced by CCM3‐silence is mediated by the activation of EphB4 forward signalling. The identified endothelial signalling pathway of CCM3‐DLL4/Notch‐EphB4‐Erk1/2 may provide an insight into mechanism of CCM3‐ablation‐mediated angiogenesis and could potentially contribute to novel therapeutic concepts for disrupting aberrant angiogenesis in CCM and in hyper‐vascularized tumours.     

Structural Mechanism of CCM3 Heterodimerization with GCKIII Kinases

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Highlights? Crystal structure of CCM3-MST4 heterodimeric complex ? Structural mechanism driving CCM3-GCKIII heterodimerization ? Conformational changes required for CCM3-GCKIII heterodimerization ? Synergistic effects of CCM3-MST4 complex on cell proliferation and migration     

Patterns of expression of the three cerebral cavernous malformation (CCM) genes during embryonic and postnatal brain development

Cerebral Cavernous Malformation (CCM) is a disease characterized by capillary-venous lesions mostly located in the central nervous system. It occurs both as a sporadic and hereditary autosomal dominant condition. Three CCM genes have been identified and shown to encode the KRIT1 (CCM1), MGC4607 (CCM2) and PDCD10 (CCM3) proteins whose functions are so far unknown. In an attempt to get some insight into the role of the 3 CCM genes, we used in situ hybridization to conduct a comparative analysis of their expression pattern at several time points during murine embryonic, postnatal and adult stages particularly within the central nervous system. A strong expression of the 3 Ccm genes was detected in the various neuronal cell layers of the brain, cerebellum and spinal cord, from embryonic to adult life. By E14.5 a moderate labelling was observed in the heart, arterial and venous large vessels with all 3 Ccm probes. Ccm2 and Ccm3 mRNAs, but not Ccm1, were clearly detected within meningeal and parenchymal cortical vessels at P8. This expression was no more detected by P19 and in adult murine brain, strongly suggesting a role for these 2 proteins in the intensive angiogenesis process occuring within the central nervous system during this period.     

A novel non-canonical mechanism of regulation of MST3 (mammalian Sterile20-related kinase 3)

The canonical pathway of regulation of the GCK (germinal centre kinase) III subgroup member, MST3 (mammalian Sterile20-related kinase 3), involves a caspase-mediated cleavage between N-terminal catalytic and C-terminal regulatory domains with possible concurrent autophosphorylation of the activation loop MST3(Thr(178)), induction of serine/threonine protein kinase activity and nuclear localization. We identified an alternative 'non-canonical' pathway of MST3 activation (regulated primarily through dephosphorylation) which may also be applicable to other GCKIII (and GCKVI) subgroup members. In the basal state, inactive MST3 co-immunoprecipitated with the Golgi protein GOLGA2/gm130 (golgin A2/Golgi matrix protein 130). Activation of MST3 by calyculin A (a protein serine/threonine phosphatase 1/2A inhibitor) stimulated (auto)phosphorylation of MST3(Thr(178)) in the catalytic domain with essentially simultaneous cis-autophosphorylation of MST3(Thr(328)) in the regulatory domain, an event also requiring the MST3(341-376) sequence which acts as a putative docking domain. MST3(Thr(178)) phosphorylation increased MST3 kinase activity, but this activity was independent of MST3(Thr(328)) phosphorylation. Interestingly, MST3(Thr(328)) lies immediately C-terminal to a STRAD (Sterile20-related adaptor) pseudokinase-like site identified recently as being involved in binding of GCKIII/GCKVI members to MO25 scaffolding proteins. MST3(Thr(178)/Thr(328)) phosphorylation was concurrent with dissociation of MST3 from GOLGA2/gm130 and association of MST3 with MO25, and MST3(Thr(328)) phosphorylation was necessary for formation of the activated MST3-MO25 holocomplex.     

PDCD10/CCM3 acts downstream of {gamma}-protocadherins to regulate neuronal survival

γ-Protocadherins (PCDH-γ) regulate neuronal survival in the vertebrate central nervous system. The molecular mechanisms of how PCDH-γ mediates this function are still not understood. In this study, we show that through their common cytoplasmic domain, different PCDH-γ isoforms interact with an intracellular adaptor protein named PDCD10 (programmed cell death 10). PDCD10 is also known as CCM3, a causative genetic defect for cerebral cavernous malformations in humans. Using RNAi-mediated knockdown, we demonstrate that PDCD10 is required for the occurrence of apoptosis upon PCDH-γ depletion in developing chicken spinal neurons. Moreover, overexpression of PDCD10 is sufficient to induce neuronal apoptosis. Taken together, our data reveal a novel function for PDCD10/CCM3, acting as a critical regulator of neuronal survival during development.     

Significance of zinc in a regulatory protein, CCM1, which regulates the carbon-concentrating mechanism in Chlamydomonas reinhardtii

In conditions with the poor availability of inorganic carbon(CO₂ and HCO₃ ^–: Ci) for photosynthesis, aquatic photosyntheticorganisms induce active Ci uptake systems that allow accumulationof Ci within the cell, the so-called carbon-concentrating mechanism(CCM). In a unicellular green alga, Chlamydomonas reinhardtii,a regulatory factor CCM1 is indispensable for the regulationof the CCM by sensing CO₂ availability. CCM1 has two putativezinc-binding domains with several conserved cysteine and histidineresidues in its N-terminal region. To determine whether thedomains actually bind zinc atoms, the N-terminal parts of CCM1were expressed as glutathione S-transferase fusion proteinsand subjected to atomic absorption spectrometry. It was foundthat 1 mol of zinc is bound to 1 mol of amino acid regions 1–71and 72–101 of CCM1, respectively. In the case of the site-directedmutant proteins, H54Y, C77V and C80V, the zinc-binding abilitywas lost. Physiological analyses of the transgenic Chlamydomonascells harboring a mutated Ccm1 gene revealed that amino acidresidues such as C36, C41, H54, C77, C80, H90 and C93 were indispensablefor induction of the CCM in response to Ci-limiting stress conditions.Size exclusion chromatography followed by immunoblot analysesindicated that CCM1 is present as a protein complex of approximately290–580 kDa independent of Ci availability.     

一中国脑海绵状血管瘤家系中发现krit1基因新的缺失突变

脑海绵状血管瘤（CCM）是多定位于中枢神经系统的一种脑部血管异常,少数在皮肤和视网膜处有并发症。依据致病基因在染色体上的不同位置分为CCM1、CCM2和CCM33种类型。目前,CCM1、CCM2和CCM3的致病基因已经被克隆,分别为krit1、MGC4607和细胞程序性死亡10基因（PDCD10）。利用连锁分析发现内蒙古的一个家系属于CCM1,突变检测发现患者CCM1基因（krit1）第9内含子和第10外显子拼接位点处存在一“GTA”缺失,该突变导致终止密码子提前出现,产生截短蛋白。实验结果支持krit1为CCM1致病基因。