Nonenzymatic domains of Kalirin7 contribute to spine morphogenesis through interactions with phosphoinositides and Abl

Like several Rho GDP/GTP exchange factors (GEFs), Kalirin7 (Kal7) contains an N-terminal Sec14 domain and multiple spectrin repeats. A natural splice variant of Kalrn lacking the Sec14 domain and four spectrin repeats is unable to increase spine formation; our goal was to understand the function of the Sec14 and spectrin repeat domains. Kal7 lacking its Sec14 domain still increased spine formation, but the spines were short. Strikingly, Kal7 truncation mutants containing only the Sec14 domain and several spectrin repeats increased spine formation. The Sec14 domain bound phosphoinositides, a minor but crucial component of cellular membranes, and binding was increased by a phosphomimetic mutation. Expression of KalSec14-GFP in nonneuronal cells impaired receptor-mediated endocytosis, linking Kal7 to membrane trafficking. Consistent with genetic studies placing Abl, a non–receptor tyrosine kinase, and the Drosophila orthologue of Kalrn into the same signaling pathway, Abl1 phosphorylated two sites in the fourth spectrin repeat of Kalirin, increasing its sensitivity to calpain-mediated degradation. Treating cortical neurons of the wild-type mouse, but not the Kal7^KO mouse, with an Abl inhibitor caused an increase in linear spine density. Phosphorylation of multiple sites in the N-terminal Sec14/spectrin region of Kal7 may allow coordination of the many signaling pathways contributing to spine morphogenesis.     

Structural organization of the nine spectrin repeats of kalirin

Sequence analysis suggests that KALRN, a Rho GDP/GTP exchange factor genetically linked to schizophrenia, could contain as many as nine tandem spectrin repeats (SRs). We expressed and purified fragments of Kalirin containing from one to five putative SRs to determine whether they formed nested structures that could endow Kalirin with the flexible rodlike properties characteristic of spectrin and dystrophin. Far-UV circular dichroism studies indicated that Kalirin contains nine SRs. On the basis of thermal denaturation, sensitivity to chemical denaturants, and the solubility of pairs of repeats, the nine SRs of Kalirin form nested structures. Modeling studies confirmed this conclusion and identified an exposed loop in SR5; consistent with the modeling, this loop was extremely labile to proteolytic cleavage. Analysis of a direpeat fragment (SR4:5) encompassing the region of Kalirin known to interact with NOS2, DISC-1, PAM, and Arf6 identified this as the least stable region. Analytical ultracentrifugation indicated that SR1:3, SR4:6, and SR7:9 were monomers and adopted an extended conformation. Gel filtration suggested that ΔKal7, a natural isoform that includes SR5:9, was monomeric and was not more extended than SR5:9. Similarly, the nine SRs of Kal7, which was also monomeric, were not more extended than SR5:9. The rigidity and flexibility of the nine SRs of Kal7, which separate its essential N-terminal Sec14p domain from its catalytic domain, play an essential role in its contribution to the formation and function of dendritic spines.     

An isoform of kalirin, a brain-specific GDP/GTP exchange factor, is enriched in the postsynaptic density fraction

Communication between membranes and the actin cytoskeleton is an important aspect of neuronal function. Regulators of actin cytoskeletal dynamics include the Rho-like small GTP-binding proteins and their exchange factors. Kalirin is a brain-specific protein, first identified through its interaction with peptidylglycine-alpha-amidating monooxygenase. In this study, we cloned rat Kalirin-7, a 7-kilobase mRNA form of Kalirin. Kalirin-7 contains nine spectrin-like repeats, a Dbl homology domain, and a pleckstrin homology domain. We found that the majority of Kalirin-7 protein is associated with synaptosomal membranes, but a fraction is cytosolic. We also detected higher molecular weight Kalirin proteins. In rat cerebral cortex, Kalirin-7 is highly enriched in the postsynaptic density fraction. In primary cultures of neurons, Kalirin-7 is detected in spine-like structures, while other forms of Kalirin are visualized in the cell soma and throughout the neurites. Kalirin-7 and its Dbl homology-pleckstrin homology domain induce formation of lamellipodia and membrane ruffling, when transiently expressed in fibroblasts, indicative of Rac1 activation. Using Rac1, the Dbl homology-pleckstrin homology domain catalyzed the in vitro exchange of bound GDP with GTP. Kalirin-7 is the first guanine-nucleotide exchange factor identified in the postsynaptic density, where it is positioned optimally to regulate signal transduction pathways connecting membrane proteins and the actin cytoskeleton.     

Identification of spectrin-like repeats required for high affinity utrophin-actin interaction

Most studies aimed at characterizing the utrophinactin interaction have focused on the amino-terminal tandem calponin homology domain. However, we recently reported evidence suggesting that spectrin-like repeats of utrophin also participate in binding to actin. Here we expressed several recombinant fragments encoding the utrophin amino-terminal domain alone or in combination with various numbers of spectrin-like repeats. We further quantitatively characterized the actin binding properties of each recombinant utrophin fragment using a high-speed sedimentation assay. To evaluate the capacity of each protein to stabilize actin filaments, we compared the effect of utrophin recombinant fragments and full-length utrophin on 6-propionyl-2-(N,N-dimethylamino)naphthalene actin depolymerization. Our results suggest that, whereas the amino-terminal domain is essential for primary interaction between utrophin and actin, spectrin-like repeats have additive effects on the affinity and stoichiometry of binding. Our data indicate that the amino-terminal domain and first 10 consecutive spectrin-like repeats recapitulate the actin binding activity of full-length utrophin more faithfully than the amino-terminal domain alone. These findings support the model for lateral association of utrophin along the actin filament and provide the molecular basis for designing the most effective utrophin "mini-genes" for treatment of dystrophinopathies.     

Identification of kalirin-7 as a potential post-synaptic density signaling hub

Kalirin-7 (Kal7), a multifunctional Rho GDP/GTP exchange factor (GEF) for Rac1 and RhoG, is embedded in the postsynaptic density at excitatory synapses, where it participates in the formation and maintenance of dendritic spines. Kal7 has been implicated in long-term potentiation, fear memories, and addiction-like behaviors. Using liquid chromatography and tandem mass spectroscopy, we identified sites phosphorylated by six PSD-localized kinases implicated in synaptic plasticity and behavior, sites phosphorylated when myc-Kal7 was expressed in non-neuronal cells and sites phosphorylated in mouse brain Kal7. A site in the Sec14p domain phosphorylated by calcium/calmodulin dependent protein kinase II, protein kinase A and protein kinase C, was phosphorylated in mouse brain but not in non-neuronal cells. Phosphorylation in the spectrin-like repeat region was more extensive in mouse brain than in non-neuronal cells, with a total of 20 sites identified. Sites in the pleckstrin homology domain and in the linker region connecting the GEF domain to the PDZ binding motif were heavily phosphorylated in both non-neuronal cells and in mouse brain and affected GEF activity. We postulate that the kinase convergence and divergence observed in Kal7 identify it as a key player in integration of the multiple inputs that regulate synaptic structure and function.     

The Sec14 homology domain regulates the cellular distribution and transforming activity of the Rho-specific guanine nucleotide exchange factor Dbs

Dbs is a Rho-specific guanine nucleotide exchange factor that was identified in a screen for proteins whose overexpression cause deregulated growth in murine fibroblasts. Dbs contains multiple recognizable motifs including a centrally located Rho-specific guanine nucleotide exchange factor domain, a COOH-terminal Src homology 3 domain, two spectrin-like repeats, and a recently identified NH(2)-terminal Sec14 homology domain. The transforming potential of Dbs is substantially activated by the removal of inhibitory sequences that lie outside of the core catalytic sequences, and in this current study we mapped this inhibition to the Sec14 domain. Surprisingly removal of the NH(2) terminus did not alter the catalytic activity of Dbs in vivo but rather altered its subcellular distribution. Whereas full-length Dbs was distributed primarily in a perinuclear structure that coincides with a marker for the Golgi apparatus, removal of the Sec14 domain was associated with translocation of Dbs to the cell periphery where it accumulated within membrane ruffles and lamellipodia. However, translocation of Dbs and the concomitant changes in the actin cytoskeleton were not sufficient to fully activate Dbs transformation. The Sec14 domain also forms intramolecular contacts with the pleckstrin homology domain, and these contacts must also be relieved to achieve full transforming activity. Collectively these observations suggest that the Sec14 domain regulates Dbs transformation through at least two distinct mechanisms, neither of which appears to directly influence the in vivo exchange activity of the protein.     

Small-Angle X-Ray Scattering Reveals Compact Domain-Domain Interactions in the N-Terminal Region of Filamin C

Filamins are multi-domain, actin cross-linking, and scaffolding proteins. In addition to the actin cross-linking function, filamins have a role in mechanosensor signaling. The mechanosensor function is mediated by domain-domain interaction in the C-terminal region of filamins. Recently, we have shown that there is a three-domain interaction module in the N-terminal region of filamins, where the neighboring domains stabilize the structure of the middle domain and thereby regulate its interaction with ligands. In this study, we have used small-angle X-ray scattering as a tool to screen for potential domain-domain interactions in the N-terminal region. We found evidence of four domain-domain interactions with varying flexibility. These results confirm our previous study showing that domains 3, 4, and 5 exist as a compact three domain module. In addition, we report interactions between domains 11–12 and 14–15, which are thus new candidate sites for mechanical regulation.     

Dystrophin and utrophin bind actin through distinct modes of contact

This study was designed to define the molecular epitopes of dystrophin-actin interaction and to directly compare the actin binding properties of dystrophin and utrophin. According to our data, dystrophin and utrophin both bound alongside actin filaments with submicromolar affinities. However, the molecular epitopes involved in actin binding differed between the two proteins. In utrophin, the amino-terminal domain and an adjacent string of the first 10 spectrin-like repeats more fully recapitulated the activities measured for full-length protein. The homologous region of dystrophin bound actin with low affinity and near 1:1 stoichiometry as previously measured for the isolated amino-terminal, tandem (CH) domain. In contrast, a dystrophin construct including a cluster of basic spectrin-like repeats and spanning from the amino terminus through repeat 17, bound actin with properties most similar to full-length dystrophin. Dystrophin and utrophin both stabilized preformed actin filaments from forced depolymerization with similar efficacies but did not appear to compete for binding sites on actin. We also found that dystrophin binding to F-actin was markedly sensitive to increasing ionic strength, although utrophin binding was unaffected. Although dystrophin and utrophin are functionally homologous actin-binding proteins, these results indicate that their respective modes of contact with actin filaments are markedly different. Finally, we reassessed the abundance of dystrophin in striated muscle using full-length protein as the standard and measured greater than 10-fold higher values than previously reported.     

Differential mechanical stability of filamin A rod segments

Prompted by recent reports suggesting that interaction of filamin A (FLNa) with its binding partners is regulated by mechanical force, we examined mechanical properties of FLNa domains using magnetic tweezers. FLNa, an actin cross-linking protein, consists of two subunits that dimerize through a C-terminal self-association domain. Each subunit contains an N-terminal spectrin-related actin-binding domain followed by 24 immunoglobulinlike (Ig) repeats. The Ig repeats in the rod 1 segment (repeats 1–15) are arranged as a linear array, whereas rod 2 (repeats 16–23) is more compact due to interdomain interactions. In the rod 1 segment, repeats 9–15 augment F-actin binding to a much greater extent than do repeats 1–8. Here, we report that the three segments are unfolded at different forces under the same loading rate. Remarkably, we found that repeats 16–23 are susceptible to forces of ∼10 pN or even less, whereas the repeats in the rod 1 segment can withstand significantly higher forces. The differential force response of FLNa Ig domains has broad implications, since these domains not only support the tension of actin network but also interact with many transmembrane and signaling proteins, mostly in the rod 2 segment. In particular, our finding of unfolding of repeats 16–23 at ∼10 pN or less is consistent with the hypothesized force-sensing function of the rod 2 segment in FLNa.     

Domain analysis of α-actinin reveals new aspects of its association with F-actin during cytokinesis

α-actinin is a rod-shaped actin cross-linking protein composed of actin binding domain, spectrin-like repeats of the central rod domain and the EF-hand domain. Cytokinesis in mammalian cells involves remodeling of equatorial actin filaments (F-actin) mediated by α-actinin. However, it remains unknown how α-actinin interacts with F-actin at the cleavage furrow. To address this question, we have conducted functional analysis of the mutant that either lacks the ability to cross-link F-actin (ABD) or to bind to F-actin (ΔABD). We found that equatorial localization of α-actinin requires both its F-actin binding and cross-linking activities. Unexpectedly, we also found that overexpression of ΔABD-GFP but not ABD-GFP frequently caused accelerated cytokinesis and ectopic furrowing similar to those observed in cells depleted of α-actinin. Immunofluorescence revealed that overexpression of ΔABD-GFP caused displacement of endogenous α-actinin and a decrease in the density of F-actin throughout the entire cortex. Biochemical experiments showed that ΔABD was able to form heterodimers with endogenous α-actinin. These results suggest that the central rod spectrin-like repeats of α-actinin is sufficient for its dimerization in vivo. Our findings uncover previously unappreciated functions of the α-actinin domains in a cell.     

SAC1-like domains of yeast SAC1, INP52, and INP53 and of human synaptojanin encode polyphosphoinositide phosphatases

The SAC1 gene product has been implicated in the regulation of actin cytoskeleton, secretion from the Golgi, and microsomal ATP transport; yet its function is unknown. Within SAC1 is an evolutionarily conserved 300-amino acid region, designated a SAC1-like domain, that is also present at the amino termini of the inositol polyphosphate 5-phosphatases, mammalian synaptojanin, and certain yeast INP5 gene products. Here we report that SAC1-like domains have intrinsic enzymatic activity that defines a new class of polyphosphoinositide phosphatase (PPIPase). Purified recombinant SAC1-like domains convert yeast lipids phosphatidylinositol (PI) 3-phosphate, PI 4-phosphate, and PI 3,5-bisphosphate to PI, whereas PI 4,5-bisphosphate is not a substrate. Yeast lacking Sac1p exhibit 10-, 2.5-, and 2-fold increases in the cellular levels of PI 4-phosphate, PI 3,5-bisphosphate, and PI 3-phosphate, respectively. The 5-phosphatase domains of synaptojanin, Inp52p, and Inp53p are also catalytic, thus representing the first examples of an inositol signaling protein with two distinct lipid phosphatase active sites within a single polypeptide chain. Together, our data provide a long sought mechanism as to how defects in Sac1p overcome certain actin mutants and bypass the requirement for yeast phosphatidylinositol/phosphatidylcholine transfer protein, Sec14p. We demonstrate that PPIPase activity is a key regulator of membrane trafficking and actin cytoskeleton organization and suggest signaling roles for phosphoinositides other than PI 4,5-bisphosphate in these processes. Additionally, the tethering of PPIPase and 5-phosphatase activities indicate a novel mechanism by which concerted phosphoinositide hydrolysis participates in membrane trafficking.     

Interaction between the triglyceride lipase ATGL and the Arf1 activator GBF1

The Arf1 exchange factor GBF1 (Golgi Brefeldin A resistance factor 1) and its effector COPI are required for delivery of ATGL (adipose triglyceride lipase) to lipid droplets (LDs). Using yeast two hybrid, co-immunoprecipitation in mammalian cells and direct protein binding approaches, we report here that GBF1 and ATGL interact directly and in cells, through multiple contact sites on each protein. The C-terminal region of ATGL interacts with N-terminal domains of GBF1, including the catalytic Sec7 domain, but not with full-length GBF1 or its entire N-terminus. The N-terminal lipase domain of ATGL (called the patatin domain) interacts with two C-terminal domains of GBF1, HDS (Homology downstream of Sec7) 1 and HDS2. These two domains of GBF1 localize to lipid droplets when expressed alone in cells, but not to the Golgi, unlike the full-length GBF1 protein, which localizes to both. We suggest that interaction of GBF1 with ATGL may be involved in the membrane trafficking pathway mediated by GBF1, Arf1 and COPI that contributes to the localization of ATGL to lipid droplets.     

Regulation of RhoGEF activity by intramolecular and intermolecular SH3 domain interactions

RhoGEFs are central controllers of small G-proteins in cells and are regulated by several mechanisms. There are at least 22 human RhoGEFs that contain SH3 domains, raising the possibility that, like several other enzymes, SH3 domains control the enzymatic activity of guanine nucleotide exchange factor (GEF) domains through intra- and/or intermolecular interactions. The structure of the N-terminal SH3 domain of Kalirin was solved using NMR spectroscopy, and it folds much like other SH3 domains. However, NMR chemical shift mapping experiments showed that this Kalirin SH3 domain is unique, containing novel cooperative binding site(s) for intramolecular PXXP ligands. Intramolecular Kalirin SH3 domain/ligand interactions, as well as binding of the Kalirin SH3 domain to the adaptor protein Crk, inhibit the GEF activity of Kalirin. This study establishes a novel molecular mechanism whereby intramolecular and intermolecular Kalirin SH3 domain/ligand interactions modulate GEF activity, a regulatory mechanism that is likely used by other RhoGEF family members.     

Dominant Negative Alleles of SEC10 Reveal Distinct Domains Involved in Secretion and Morphogenesis in Yeast

The accurate targeting of secretory vesicles to distinct sites on the plasma membrane is necessary to achieve polarized growth and to establish specialized domains at the surface of eukaryotic cells. Members of a protein complex required for exocytosis, the exocyst, have been localized to regions of active secretion in the budding yeast Saccharomyces cerevisiae where they may function to specify sites on the plasma membrane for vesicle docking and fusion. In this study we have addressed the function of one member of the exocyst complex, Sec10p. We have identified two functional domains of Sec10p that act in a dominant-negative manner to inhibit cell growth upon overexpression. Phenotypic and biochemical analysis of the dominant-negative mutants points to a bifunctional role for Sec10p. One domain, consisting of the amino-terminal two-thirds of Sec10p directly interacts with Sec15p, another exocyst component. Overexpression of this domain displaces the full-length Sec10 from the exocyst complex, resulting in a block in exocytosis and an accumulation of secretory vesicles. The carboxy-terminal domain of Sec10p does not interact with other members of the exocyst complex and expression of this domain does not cause a secretory defect. Rather, this mutant results in the formation of elongated cells, suggesting that the second domain of Sec10p is required for morphogenesis, perhaps regulating the reorientation of the secretory pathway from the tip of the emerging daughter cell toward the mother–daughter connection during cell cycle progression.     

Brefeldin A inhibited activity of the sec7 domain of p200, a mammalian guanine nucleotide-exchange protein for ADP-ribosylation factors.

A brefeldin A (BFA)-inhibited guanine nucleotide-exchange protein (GEP) for ADP-ribosylation factors (ARF) was purified earlier from bovine brain cytosol. Cloning and expression of the cDNA confirmed that the recombinant protein (p200) is a BFA-sensitive ARF GEP. p200 contains a domain that is 50% identical in amino acid sequence to a region in yeast Sec7, termed the Sec7 domain. Sec7 domains have been identified also in other proteins with ARF GEP activity, some of which are not inhibited by BFA. To identify structural elements that influence GEP activity and its BFA sensitivity, several truncated mutants of p200 were made. Deletion of sequence C-terminal to the Sec7 domain did not affect GEP activity. A protein lacking 594 amino acids at the N terminus, as well as sequence following the Sec7 domain, also had high activity. The mutant lacking 630 N-terminal amino acids was, however, only 1% as active, as was the Sec7 domain itself (mutant lacking 697 N-terminal residues). It appears that the Sec7 domain of p200 contains the catalytic site but additional sequence (perhaps especially that between positions 595 and 630) modifies activity dramatically. Myristoylated recombinant ARFs were better than non-myristoylated as substrates; ARFs 1 and 3 were better than ARF5, and no activity was detected with ARF6. Physical interaction of the Sec7 domain with an ARF1 mutant was demonstrated, but it was much weaker than that of the cytohesin-1 Sec7 domain with the same ARF protein. Effects of BFA on p200 and all mutants with high activity were similar with approximately 50% inhibition at 相似文献   

The Unique Non-Catalytic C-Terminus of P37delta-PI3K Adds Proliferative Properties In Vitro and In Vivo

The PI3K/Akt pathway is central for numerous cellular functions and is frequently deregulated in human cancers. The catalytic subunits of PI3K, p110, are thought to have a potential oncogenic function, and the regulatory subunit p85 exerts tumor suppressor properties. The fruit fly, Drosophila melanogaster, is a highly suitable system to investigate PI3K signaling, expressing one catalytic, Dp110, and one regulatory subunit, Dp60, and both show strong homology with the human PI3K proteins p110 and p85. We recently showed that p37δ, an alternatively spliced product of human PI3K p110δ, displayed strong proliferation-promoting properties despite lacking the catalytic domain completely. Here we functionally evaluate the different domains of human p37δ in Drosophila. The N-terminal region of Dp110 alone promotes cell proliferation, and we show that the unique C-terminal region of human p37δ further enhances these proliferative properties, both when expressed in Drosophila, and in human HEK-293 cells. Surprisingly, although the N-terminal region of Dp110 and the C-terminal region of p37δ both display proliferative effects, over-expression of full length Dp110 or the N-terminal part of Dp110 decreases survival in Drosophila, whereas the unique C-terminal region of p37δ prevents this effect. Furthermore, we found that the N-terminal region of the catalytic subunit of PI3K p110, including only the Dp60 (p85)-binding domain and a minor part of the Ras binding domain, rescues phenotypes with severely impaired development caused by Dp60 over-expression in Drosophila, possibly by regulating the levels of Dp60, and also by increasing the levels of phosphorylated Akt. Our results indicate a novel kinase-independent function of the PI3K catalytic subunit.     

The human formin FHOD1 contains a bipartite structure of FH3 and GTPase-binding domains required for activation

Formins induce the nucleation and polymerization of unbranched actin filaments. They share three homology domains required for profilin binding, actin polymerization, and regulation. Diaphanous-related formins (DRFs) are activated by GTPases of the Rho/Rac family, whose interaction with the N-terminal formin domain is thought to displace a C-terminal Diaphanous-autoregulatory domain (DAD). We have determined the structure of the N-terminal domains of FHOD1 consisting of a GTPase-binding domain (GBD) and the DAD-recognition domain FH3. In contrast to the formin mDia1, the FHOD1-GBD reveals a ubiquitin superfold as found similarly in c-Raf1 or PI3 kinase. This GBD is recruited by Rac and Ras GTPases in cells and plays an essential role for FHOD1-mediated actin remodeling. The FHOD1-FH3 domain is composed of five armadillo repeats, similarly to other formins. Mutation of one residue in the predicted DAD-interaction surface efficiently activates FHOD1 in cells. These results demonstrate that DRFs have evolved different molecular solutions to govern their autoregulation and GTPase specificity.