Striatin, a calmodulin-dependent scaffolding protein, directly binds caveolin-1.

Caveolins are scaffolding proteins able to collect on caveolae a large number of signalling proteins bearing a caveolin-binding motif. The proteins of the striatin family, striatin, SG2NA, and zinedin, are composed of several conserved, collinearly aligned, protein-protein association domains, among which a putative caveolin-binding domain [Castets et al. (2000) J. Biol. Chem. 275, 19970-19977]. They are associated in part with membranes. These proteins are mainly expressed within neurons and thought to act both as scaffolds and as Ca(2+)-dependent signalling proteins [Bartoli et al. (1999) J. Neurobiol. 40, 234-243]. Here, we show that (1) rat brain striatin, SG2NA and zinedin co-immunoprecipitate with caveolin-1; (2) all are pulled down by glutathione-S-transferase (GST)-caveolin-1; (3) a fragment of recombinant striatin containing the putative caveolin-binding domain binds GST-caveolin-1. Hence, it is likely that the proteins of the striatin family are addressed to membrane microdomains by their binding to caveolin, in accordance with their putative role in membrane trafficking [Baillat et al. (2001) Mol. Biol. Cell 12, 663-673].     

Expression and distribution of phocein and members of the striatin family in neurones of rat peripheral ganglia

Phocein and members of the striatin family (striatin, SG2NA and zinedin) are intracellular proteins, mainly expressed in neurones of the mammalian central nervous system where they are thought to be involved in vesicular traffic and Ca(2+) signalling. Here, we have investigated whether these proteins are also present in the peripheral nervous system, by analysing their expression and distribution within sensory neurones of the vagal (nodose and jugular) ganglia, the petrosal ganglion, the dorsal root ganglion, and also in the sympathetic neurones of the superior cervical ganglion. RT-PCR experiments showed that mRNAs of phocein, striatin, SG2NA and zinedin are present in all studied peripheral ganglia. Immunocytochemical detections demonstrate that phocein, striatin and SG2NA are expressed in neurones of vagal, petrosal and dorsal root ganglia. Immunoblotting experiments confirm these data and in addition demonstrate that: (1) the proteins phocein, striatin and SG2NA are also present in the superior cervical ganglion and (2) zinedin is detected in all studied ganglia. The distribution appears to differ: immunoreactivity for striatin and SG2NA is found only in soma of sensory neurons, whereas immunoreactivity for phocein is observed in both soma and processes. Our study thus demonstrates that phocein and the members of the striatin family are expressed not only in central nervous system but also in the peripheral nervous system and, in particular, in afferent sensory neurones.     

Targeting of proteins of the striatin family to dendritic spines: role of the coiled-coil domain

Striatin, SG2NA and zinedin, the three mammalian members of the striatin family are multimodular WD-repeat, calmodulin and calveolin-binding proteins. These scaffolding proteins, involved in both signaling and trafficking, are highly expressed in neurons. Using ultrastructural immunolabeling, we showed that, in Purkinje cells and hippocampal neurons, SG2NA is confined to the somatodendritic compartment with the highest density in dendritic spines. In cultured hippocampal neurons, SG2NA is also highly concentrated in dendritic spines. By expressing truncated forms of HA-tagged SG2NAbeta, we demonstrated that the coiled-coil domain plays an essential role in the targeting of SG2NA within spines. Furthermore, co-immunoprecipitation experiments indicate that this coiled-coil domain is also crucial for the homo- and hetero-oligomerization of these proteins. Thus, oligomerization of the striatin family proteins is probably an obligatory step for their routing to the dendritic spines, and hetero-oligomerization explains why all these proteins are often co-expressed in the neurons of the rat brain and spinal cord.     

The Colletotrichum graminicola striatin orthologue Str1 is necessary for anastomosis and is a virulence factor

Striatin family proteins are key regulators in signalling pathways in fungi and animals. These scaffold proteins contain four conserved domains: a caveolin‐binding domain, a coiled‐coil motif and a calmodulin‐binding domain at the N‐terminus, and a WD‐repeat domain at the C‐terminus. Fungal striatin orthologues are associated with sexual development, hyphal growth and plant pathogenesis. In Fusarium verticillioides, the striatin orthologue Fsr1 promotes virulence in the maize stalk. The relationship between fungal striatins and pathogenicity remains largely unexplored. In this study, we demonstrate that the Colletotrichum graminicola striatin orthologue Str1 is required for full stalk rot and leaf blight virulence in maize. Pathogenicity assays show that the striatin mutant strain (Δstr1) produces functional appressoria, but infection and colonization are attenuated. Additional phenotypes of the Δstr1 mutant include reduced radial growth and compromised hyphal fusion. In comparison with the wild‐type, Δstr1 also shows a defect in sexual development and produces fewer and shorter conidia. Together with the fact that F. verticillioides fsr1 can complement Δstr1, our results indicate that C. graminicola Str1 shares five phenotypes with striatin orthologues in other fungal species: hyphal growth, hyphal fusion, conidiation, sexual development and virulence. We propose that fungal striatins, like mammalian striatins, act as scaffolding molecules that cross‐link multiple signal transduction pathways.     

Distribution of zinedin in the rat brain

Members of the striatin family are scaffolding proteins involved in numerous signaling pathways principally in neurons. Zinedin is the only member of this protein family for which the brain distribution has not been determined so far. Here, we have validated a specific antibody against zinedin and used this tool to study the localization of zinedin at cellular and sub-cellular levels in the rat brain. Zinedin is primarily expressed in neurons of the hippocampus, cerebral cortex, olfactory bulb and caudate putamen nucleus. Like other members of the striatin family, zinedin displays a polarized distribution in the somato-dendritic compartment of neurons and is enriched in dendritic spines. The rostral expression of zinedin as well as its compartmented distribution in dendritic spines may have important implications not only for zinedin function but also in the physiology of dendritic spines of a particular subset of neurons.     

WD40 repeat proteins striatin and S/G(2) nuclear autoantigen are members of a novel family of calmodulin-binding proteins that associate with protein phosphatase 2A

Protein phosphatase 2A (PP2A) is a multifunctional serine/threonine phosphatase that is critical to many cellular processes including development, neuronal signaling, cell cycle regulation, and viral transformation. PP2A has been implicated in Ca(2+)-dependent signaling pathways, but how PP2A is targeted to these pathways is not understood. We have identified two calmodulin (CaM)-binding proteins that form stable complexes with the PP2A A/C heterodimer and may represent a novel family of PP2A B-type subunits. These two proteins, striatin and S/G(2) nuclear autoantigen (SG2NA), are highly related WD40 repeat proteins of previously unknown function and distinct subcellular localizations. Striatin has been reported to associate with the post-synaptic densities of neurons, whereas SG2NA has been reported to be a nuclear protein expressed primarily during the S and G(2) phases of the cell cycle. We show that SG2NA, like striatin, binds to CaM in a Ca(2+)-dependent manner. In addition to CaM and PP2A, several unidentified proteins stably associate with the striatin-PP2A and SG2NA-PP2A complexes. Thus, one mechanism of targeting and organizing PP2A with components of Ca(2+)-dependent signaling pathways may be through the molecular scaffolding proteins striatin and SG2NA.     

Molecular cloning and characterization of phocein, a protein found from the Golgi complex to dendritic spines

Phocein is a widely expressed, highly conserved intracellular protein of 225 amino acids, the sequence of which has limited homology to the sigma subunits from clathrin adaptor complexes and contains an additional stretch bearing a putative SH3-binding domain. This sequence is evolutionarily very conserved (80% identity between Drosophila melanogaster and human). Phocein was discovered by a yeast two-hybrid screen using striatin as a bait. Striatin, SG2NA, and zinedin, the three mammalian members of the striatin family, are multimodular, WD-repeat, and calmodulin-binding proteins. The interaction of phocein with striatin, SG2NA, and zinedin was validated in vitro by coimmunoprecipitation and pull-down experiments. Fractionation of brain and HeLa cells showed that phocein is associated with membranes, as well as present in the cytosol where it behaves as a protein complex. The molecular interaction between SG2NA and phocein was confirmed by their in vivo colocalization, as observed in HeLa cells where antibodies directed against either phocein or SG2NA immunostained the Golgi complex. A 2-min brefeldin A treatment of HeLa cells induced the redistribution of both proteins. Immunocytochemical studies of adult rat brain sections showed that phocein reactivity, present in many types of neurons, is strictly somato-dendritic and extends down to spines, just as do striatin and SG2NA.     

Zinedin, SG2NA, and striatin are calmodulin-binding, WD repeat proteins principally expressed in the brain

Striatin is an intracellular protein characterized by four protein-protein interaction domains, a caveolin-binding motif, a coiled-coil structure, a calmodulin-binding domain, and a WD repeat domain, suggesting that it is a signaling or a scaffold protein. Down-regulation of striatin, which is expressed in a few subsets of neurons, impairs the growth of dendrites as well as rat locomotor activity (Bartoli, M., Ternaux, J. P., Forni, C., Portalier, P., Salin, P., Amalric, M., and Monneron, A. (1999) J. Neurobiol. 40, 234-243). Zinedin, a "novel" protein described here, and SG2NA share with striatin identical protein-protein interaction domains and the same overall domain structure. A phylogenetic analysis supports the hypothesis that they constitute a multigenic family deriving from an ancestral gene. DNA probes and antibodies raised against specific domains of each protein showed that zinedin is mainly expressed in the central nervous system, whereas SG2NA, of more widespread occurrence, is mainly expressed in the brain and muscle. All three proteins are both cytosolic and membrane-bound. All three bind calmodulin in the presence of Ca(2+). In rat brain, SG2NA and striatin are generally not found in the same neurons. Both localize to the soma and dendrites, suggesting that they share a similar type of addressing and closely related functions.     

Fsr1, a striatin homologue,forms an endomembrane‐associated complex that regulates virulence in the maize pathogen Fusarium verticillioides

Fsr1, a homologue of mammalian striatin, containing multiple protein‐binding domains and a coiled‐coil (CC) domain, is critical for Fusarium verticillioides virulence. In mammals, striatin interacts with multiple proteins to form a STRIPAK (striatin‐interacting phosphatase and kinase) complex that regulates a variety of developmental processes and cellular mechanisms. In this study, we identified the homologue of a key mammalian STRIPAK component STRIP1/2 (striatin‐interacting proteins 1 and 2) in F. verticillioides, FvStp1, which interacts with Fsr1 in vivo. Gene deletion analysis indicates that FvStp1 is critical for F. verticillioides stalk rot virulence. In addition, we identified three proteins, designated FvCyp1, FvScp1 and FvSel1, which interact with the Fsr1 CC domain via a yeast two‐hybrid screen. Importantly, FvCyp1, FvScp1 and FvSel1 co‐localize to endomembrane structures, each having a preferred localization in the cell, and they are all required for F. verticillioides stalk rot virulence. Moreover, these proteins are necessary for the correct localization of Fsr1 to the endoplasmic reticulum (ER) and nuclear envelope. Thus, we identified several novel components in the STRIPAK complex that regulates F. verticillioides virulence, and propose that the correct organization and localization of Fsr1 are critical for STRIPAK complex function.     

A novel calmodulin-binding protein, belonging to the WD-repeat family, is localized in dendrites of a subset of CNS neurons

A rat brain synaptosomal protein of 110,000 M(r) present in a fraction highly enriched in adenylyl cyclase activity was microsequenced (Castets, F., G. Baillat, S. Mirzoeva, K. Mabrouk, J. Garin, J. d'Alayer, and A. Monneron. 1994. Biochemistry. 33:5063-5069). Peptide sequences were used to clone a cDNA encoding a novel, 780-amino acid protein named striatin. Striatin is a member of the WD-repeat family (Neer, E.J., C.J. Schmidt, R. Nambudripad, and T.F. Smith. 1994. Nature (Lond.). 371:297-300), the first one known to bind calmodulin (CaM) in the presence of Ca++. Subcellular fractionation shows that striatin is a membrane-associated, Lubrol-soluble protein. As analyzed by Northern blots, in situ hybridization, and immunocytochemistry, striatin is localized in the central nervous system, where it is confined to a subset of neurons, many of which are associated with the motor system. In particular, striatin is conspicuous in the dorsal part of the striatum, as well as in motoneurons. Furthermore, striatin is essentially found in dendrites, but not in axons, and is most abundant in dendritic spines. We propose that striatin interacts, through its WD- repeat domain and in a CaM/Ca(++)-dependent manner, with one or several members of a surrounding cluster of molecules engaged in a Ca(++)- signaling pathway specific to excitatory synapses.     

A mammalian homolog of yeast MOB1 is both a member and a putative substrate of striatin family-protein phosphatase 2A complexes

Striatin and S/G(2) nuclear autoantigen (SG2NA) are related proteins that contain membrane binding domains and associate with protein phosphatase 2A (PP2A) and many additional proteins that may be PP2A regulatory targets. Here we identify a major member of these complexes as class II mMOB1, a mammalian homolog of the yeast protein MOB1, and show that its phosphorylation appears to be regulated by PP2A. Yeast MOB1 is critical for cytoskeletal reorganization during cytokinesis and exit from mitosis. We show that mMOB1 associated with PP2A is not detectably phosphorylated in asynchronous murine fibroblasts. However, treatment with the PP2A inhibitor okadaic acid induces phosphorylation of PP2A-associated mMOB1 on serine. Moreover, specific inhibition of PP2A also results in hyperphosphorylation of striatin, SG2NA, and three unidentified proteins, suggesting that these proteins may also be regulated by PP2A. Indirect immunofluorescence produced highly similar staining patterns for striatin, SG2NA, and mMOB1, with the highest concentrations for each protein adjacent to the nuclear membrane. We also present evidence that these complexes may interact with each other. These data are consistent with a model in which PP2A may regulate mMOB1, striatin, and SG2NA to modulate changes in the cytoskeleton or interactions between the cytoskeleton and membrane structures.     

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.     

A WD40 repeat protein regulates fungal cell differentiation and can be replaced functionally by the mammalian homologue striatin

Fruiting body development in fungi is a complex cellular differentiation process that is controlled by more than 100 developmental genes. Mutants of the filamentous fungus Sordaria macrospora showing defects in fruiting body formation are pertinent sources for the identification of components of this multicellular differentiation process. Here we show that the sterile mutant pro11 carries a defect in the pro11 gene encoding a multimodular WD40 repeat protein. Complementation analysis indicates that the wild-type gene or C-terminally truncated versions of the wild-type protein are able to restore the fertile phenotype in mutant pro11. PRO11 shows significant homology to several vertebrate WD40 proteins, such as striatin and zinedin, which seem to be involved in Ca²⁺-dependent signaling in cells of the central nervous system and are supposed to function as scaffolding proteins linking signaling and eukaryotic endocytosis. Cloning of a mouse cDNA encoding striatin allowed functional substitution of the wild-type protein with restoration of fertility in mutant pro11. Our data strongly suggest that an evolutionarily conserved cellular process controlling eukaryotic cell differentiation may regulate fruiting body formation.     

Down-regulation of striatin, a neuronal calmodulin-binding protein, impairs rat locomotor activity.

Striatin, an intraneuronal, calmodulin-binding protein addressed to dendrites and spines, is expressed in the motor system, particularly the striatum and motoneurons. Striatin contains a high number of domains mediating protein-protein interactions, suggesting a role within a dendritic Ca(2+)-signaling pathway. Here, we explored the hypothesis of a direct role of striatin in the motor control of behaving rats, by using an antisense strategy based on oligodeoxynucleotides (ODN). Rats were treated by intracerebroventricular infusion of a striatin antisense ODN (A-ODN) or mismatch ODN (M-ODN) delivered by osmotic pumps over 6 days. A significant decrease in the nocturnal locomotor activity of A-ODN-treated rats was observed after 5 days of treatment. Hypomotricity was correlated with a 60% decrease in striatin content of the striata of A-ODN-treated rats sacrificed on day 6. Striatin thus plays a role in the control of motor function. To approach the cellular mechanisms in which striatin is involved, striatin down-regulation was studied in a comparatively simpler model: purified rat spinal motoneurons which retain their polarity in culture. Treatment of cells by the striatin A-ODN resulted in the impairement of the growth of dendrites but not axon. The decrease in dendritic growth paralleled the loss of striatin. This model allows analysis of the molecular basis of striatin function in the dynamic changes occurring in growing dendrites, and offers clues to unravel its function within spines.     

Down‐regulation of striatin,a neuronal calmodulin‐binding protein,impairs rat locomotor activity

Striatin, an intraneuronal, calmodulin‐binding protein addressed to dendrites and spines, is expressed in the motor system, particularly the striatum and motoneurons. Striatin contains a high number of domains mediating protein–protein interactions, suggesting a role within a dendritic Ca²⁺‐signaling pathway. Here, we explored the hypothesis of a direct role of striatin in the motor control of behaving rats, by using an antisense strategy based on oligodeoxynucleotides (ODN). Rats were treated by intracerebroventricular infusion of a striatin antisense ODN (A‐ODN) or mismatch ODN (M‐ODN) delivered by osmotic pumps over 6 days. A significant decrease in the nocturnal locomotor activity of A‐ODN–treated rats was observed after 5 days of treatment. Hypomotricity was correlated with a 60% decrease in striatin content of the striata of A‐ODN–treated rats sacrificed on day 6. Striatin thus plays a role in the control of motor function. To approach the cellular mechanisms in which striatin is involved, striatin down‐regulation was studied in a comparatively simpler model: purified rat spinal motoneurons which retain their polarity in culture. Treatment of cells by the striatin A‐ODN resulted in the impairement of the growth of dendrites but not axon. The decrease in dendritic growth paralleled the loss of striatin. This model allows analysis of the molecular basis of striatin function in the dynamic changes occurring in growing dendrites, and offers clues to unravel its function within spines. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 234–243, 1999     

A homologue of the human STRIPAK complex controls sexual development in fungi

Sexual development in fungi is a complex process involving the generation of new cell types and tissues - an essential step for all eukaryotic life. The characterization of sterile mutants in the ascomycete Sordaria macrospora has led to a number of proteins involved in sexual development, but a link between these proteins is still missing. Using a combined tandem-affinity purification/mass spectrometry approach, we showed in vivo association of developmental protein PRO22 with PRO11, homologue of mammalian striatin, and SmPP2AA, scaffolding subunit of protein phosphatase 2A. Further experiments extended the protein network to the putative kinase activator SmMOB3, known to be involved in sexual development. Extensive yeast two-hybrid studies allowed us to pinpoint functional domains involved in protein-protein interaction. We show for the first time that a number of already known factors together with new components associate in vivo to form a highly conserved multi-subunit complex. Strikingly, a similar complex has been described in humans, but the function of this so-called striatin interacting phosphatase and kinase (STRIPAK) complex is largely unknown. In S. macrospora, truncation of PRO11 and PRO22 leads to distinct defects in sexual development and cell fusion, indicating a role for the fungal STRIPAK complex in both processes.     

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.     

WD-40 repeat protein SG2NA has multiple splice variants with tissue restricted and growth responsive properties

SG2NA is a member of the striatin family of WD-40 repeat proteins with potential scaffolding functions. It was originally identified as a tumor antigen with increased expression during S to G2 phase of cell cycle. We report here that mouse SG2NA has at least five novel splice variants of which two are devoid of the carboxyl terminal WD-40 repeats. The variants of SG2NA are generated by alternative splicing at the exon 7-9 regions and differ in their expression profiles in various tissues tested. While the 83, 78, 38 and 35 kDa variants are present in both brain and heart, the 87 kDa form is brain specific. Also, the expression of 35 kDa variant is more in neonatal than in adult tissues. Western analysis suggests that the SG2NA isoforms differentially respond to growth stimuli. Upon serum stimulation, while the 35 kDa variant is increased, the 78 kDa form is diminished. Splicing variation of SG2NA is conserved in metazoan evolution. In embryonic chicken there are at least four variants of which one is present in brain but absent in heart. Taken together, splicing variation of SG2NA might have some critical roles in differentiation and maturation in metazoan cells.     

Methylation of the protein phosphatase 2A catalytic subunit is essential for association of Balpha regulatory subunit but not SG2NA, striatin, or polyomavirus middle tumor antigen

Binding of different regulatory subunits and methylation of the catalytic (C) subunit carboxy-terminal leucine 309 are two important mechanisms by which protein phosphatase 2A (PP2A) can be regulated. In this study, both genetic and biochemical approaches were used to investigate regulation of regulatory subunit binding by C subunit methylation. Monoclonal antibodies selectively recognizing unmethylated C subunit were used to quantitate the methylation status of wild-type and mutant C subunits. Analysis of 13 C subunit mutants showed that both carboxy-terminal and active site residues are important for maintaining methylation in vivo. Severe impairment of methylation invariably led to a dramatic decrease in Balpha subunit binding but not of striatin, SG2NA, or polyomavirus middle tumor antigen (MT) binding. In fact, most unmethylated C subunit mutants showed enhanced binding to striatin and SG2NA. Certain carboxy-terminal mutations decreased Balpha subunit binding without greatly affecting methylation, indicating that Balpha subunit binding is not required for a high steady-state level of C subunit methylation. Demethylation of PP2A in cell lysates with recombinant PP2A methylesterase greatly decreased the amount of C subunit that could be coimmunoprecipitated via the Balpha subunit but not the amount that could be coimmunoprecipitated with Aalpha subunit or MT. When C subunit methylation levels were greatly reduced in vivo, Balpha subunits were found complexed exclusively to methylated C subunits, whereas striatin and SG2NA in the same cells bound both methylated and unmethylated C subunits. Thus, C subunit methylation is critical for assembly of PP2A heterotrimers containing Balpha subunit but not for formation of heterotrimers containing MT, striatin, or SG2NA. These findings suggest that methylation may be able to selectively regulate the association of certain regulatory subunits with the A/C heterodimer.