PIX Proteins Research Articles

The Rho-GEF PIX-1 directs assembly or stability of lateral attachment structures between muscle cells

PIX proteins are guanine nucleotide exchange factors (GEFs) that activate Rac and Cdc42, and are known to have numerous functions in various cell types. Here, we show that a PIX protein has an important function in muscle. From a genetic screen in C. elegans, we found that pix-1 is required for the assembly of integrin adhesion complexes (IACs) at borders between muscle cells, and is required for locomotion of the animal. A pix-1 null mutant has a reduced level of activated Rac in muscle. PIX-1 localizes to IACs at muscle cell boundaries, M-lines and dense bodies. Mutations in genes encoding proteins at known steps of the PIX signaling pathway show defects at muscle cell boundaries. A missense mutation in a highly conserved residue in the RacGEF domain results in normal levels of PIX-1 protein, but a reduced level of activated Rac in muscle, and abnormal IACs at muscle cell boundaries.

Regions of bovine adenovirus-3 IVa2 involved in nuclear/nucleolar localization and interaction with pV

Bovine adenovirus-3 (BAdV-3) is a non enveloped, icosahedral DNA virus containing a genome of 34446 bps. The intermediate region of BAdV-3 encodes pIX and IVa2 proteins. Here, we report the characterization of BAdV-3 IVa2. Anti-IVa2 serum detected a 50 kDa protein at 24–48 h post infection in BAdV-3 infected cells. The IVa2 localizes to nucleus and nucleolus of BAdV-3 infected cells. Analysis of mutant IVa2 demonstrated that amino acids 1–25 and 373–448 are required for nuclear and nucleolar localization of IVa2, respectively. The nuclear import of IVa2 utilize importin α -1 of importin nuclear import pathway. Although deletion/substitution of amino acids 4–18 is sufficient to abrogate the nuclear localization of IVa2, amino acids 1–25 are required for nuclear localization of a cytoplasmic protein. Furthermore, we demonstrate that amino acids 1–25 and 120–140 of IVa2 interact with importin α-1 and pV proteins, respectively in BAdV-3 infected cells.

Expanding functions of GIT Arf GTPase-activating proteins, PIX Rho guanine nucleotide exchange factors and GIT-PIX complexes.

The GIT proteins, GIT1 and GIT2, are GTPase-activating proteins (inactivators) for the ADP-ribosylation factor (Arf) small GTP-binding proteins, and function to limit the activity of Arf proteins. The PIX proteins, α-PIX and β-PIX (also known as ARHGEF6 and ARHGEF7, respectively), are guanine nucleotide exchange factors (activators) for the Rho family small GTP-binding protein family members Rac1 and Cdc42. Through their multi-domain structures, GIT and PIX proteins can also function as signaling scaffolds by binding to numerous protein partners. Importantly, the constitutive association of GIT and PIX proteins into oligomeric GIT-PIX complexes allows these two proteins to function together as subunits of a larger structure that coordinates two distinct small GTP-binding protein pathways and serves as multivalent scaffold for the partners of both constituent subunits. Studies have revealed the involvement of GIT and PIX proteins, and of the GIT-PIX complex, in numerous fundamental cellular processes through a wide variety of mechanisms, pathways and signaling partners. In this Commentary, we discuss recent findings in key physiological systems that exemplify current understanding of the function of this important regulatory complex. Further, we draw attention to gaps in crucial information that remain to be filled to allow a better understanding of the many roles of the GIT-PIX complex in health and disease.

A Critical Role of GIT1 in Vertebrate and Invertebrate Brain Development.

GIT1, a multifunctional signaling adaptor protein, is implicated in the development of dendritic spines and neuronal synapses. GIT1 forms a signaling complex with PIX, RAC, and PAK proteins that is known to play important roles in brain development. Here we found that Git1-knockout (Git1-/-) mice show a microcephaly-like small brain phenotype, which appears to be caused by reduced neuronal size rather than number. Git1-/- mice also show decreased dendritic spine number without morphological alterations in the hippocampus. Behaviorally, Git1-/- mice show impaired motor coordination and learning and memory. In addition, adult dGit Drosophila mutants show decreased brain size and abnormal morphology of the mushroom body. These results suggest that GIT1 is important for brain development in both rodents and flies.

Pak1 regulates the orientation of apical polarization and lumen formation by distinct pathways.

The development of the basic architecture of branching tubules enclosing a central lumen that characterizes most epithelial organs crucially depends on the apico-basolateral polarization of epithelial cells. Signals from the extracellular matrix control the orientation of the apical surface, so that it faces the lumen interior, opposite to cell-matrix adhesion sites. This orientation of the apical surface is thought to be intrinsically linked to the formation of single lumens. We previously demonstrated in three-dimensional cyst cultures of Madin-Darby canine kidney (MDCK) cells that signaling by β1 integrins regulates the orientation of the apical surface, via a mechanism that depends on the activity of the small GTPase Rac1. Here, we investigated whether the Rac1 effector Pak1 is a downstream effector in this pathway. Expression of constitutive active Pak1 phenocopies the effect of β1 integrin inhibition in that it misorients the apical surface and induces a multilumen phenotype. The misorientation of apical surfaces depends on the interaction of active Pak1 with PIX proteins and is linked to defects in basement membrane assembly. In contrast, the multilumen phenotype was independent of PIX and the basement membrane. Therefore, Pak1 likely regulates apical polarization and lumen formation by two distinct pathways.

Biochemical and functional characterisation of αPIX, a specific regulator of axonal and dendritic branching in hippocampal neurons

PIX proteins are exchange factors for Rac and Cdc42 GTPases that are differentially expressed in the brain, where they are implicated in neuronal morphogenesis. The PIX family includes the two members αPIX and βPIX, and the gene of αPIX is mutated in patients with intellectual disability. We have analysed the expression of PIX proteins in the developing brain and addressed their role during early hippocampal neuron development. Mass spectrometry identified several βPIX isoforms and a major p75 αPIX isoform in brain and hippocampal cultures. PIX proteins expression increased with time during neuronal differentiation in vitro. The PIX partners GIT1 and GIT2 are also found in brain and their expression was increased during neuronal differentiation. We found that αPIX, but not βPIX, was required for proper hippocampal neuron differentiation, since silencing of αPIX specifically hampered dendritogenesis and axonal branching. Interestingly, the depletion of GIT2 but not GIT1 mimicked the phenotype observed after αPIX knock-down. Over-expression of αPIX specifically enhanced dendritic branching, while both αPIX and βPIX over-expression affected axonal morphology. Again, only over-expression of GIT2, but not GIT1, affected neuritic morphology. The results indicate that αPIX and GIT2 are required for neuronal differentiation, and suggest that they are part of the same pathway, while GIT1 and βPIX are dispensable for early hippocampal neurons development.

Abstract 3422: N-α-acetyltransferase 10 protein suppresses cancer cell metastasis by binding PIX proteins and inhibiting Cdc42/Rac1 activity

Abstract 80%-90% of eukaryotic proteins are N-α-terminally acetylated. They are mediated by specific N-α-acetyltransferases. However, the biological importance of these N-α-acetyltransferases remains largely unknown. Recently, these N-α-acetyltransferases have been recognized as important characters in many biological processes, including embryonic development, neuron disease and cancer. N-α-acetyltransferase A (Nat A) was first characterized in yeast, it composed of Naa10p, also known as hARD1 (human Arrest defective 1), and Naa15p, also known as NATH (N-acetyl transferase human), are responsible for the N-α-acetylation of majority proteins in mammalian cells. Naa10p is known to be involved in cell cycle control and apoptosis. However, the role of Naa10p in human cancers is elusive. In this study, we found that elevated Naa10p levels are associated with less metastasis to lymph nodes and better prognosis of cancer patients. Naa10p significantly suppressed migration, tumor growth, and metastasis independent of its enzymatic activity. Instead, Naa10p binds to the GIT-binding domain of PIX, thereby preventing the formation of the GIT-PIX-Paxillin complex, resulting in reduced intrinsic Cdc42/Rac1 activity and decreased cell migration. Forced expression of PIX in Naa10-transfected tumor cells restored the migration and metastasis ability. We suggest that Naa10p functions as a tumor metastasis suppressor by disrupting the migratory complex, PIX-GIT- Paxillin, in cancer cells. Our study not only highlights the potential of Naa10p as a therapeutic target for tumor metastasis, but also identifies Naa10p as a regulatory component of the migration complex PIX-GIT-Paxillin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3422. doi:1538-7445.AM2012-3422

N-α-Acetyltransferase 10 Protein Suppresses Cancer Cell Metastasis by Binding PIX Proteins and Inhibiting Cdc42/Rac1 Activity

N-α-acetyltransferase 10 protein, Naa10p, is an N-acetyltransferase known to be involved in cell cycle control. We found that Naa10p was expressed lower in varieties of malignancies with lymph node metastasis compared with non-lymph node metastasis. Higher Naa10p expression correlates the survival of lung cancer patients. Naa10p significantly suppressed migration, tumor growth, and metastasis independent of its enzymatic activity. Instead, Naa10p binds to the GIT-binding domain of PIX, thereby preventing the formation of the GIT-PIX-Paxillin complex, resulting in reduced intrinsic Cdc42/Rac1 activity and decreased cell migration. Forced expression of PIX in Naa10-transfected tumor cells restored the migration and metastasis ability. We suggest that Naa10p functions as a tumor metastasis suppressor by disrupting the migratory complex, PIX-GIT- Paxillin, in cancer cells.

Dissecting Activation of the PAK1 Kinase at Protrusions in Living Cells

The p21-activated kinase (PAK) 1 kinase, an effector of the Cdc42 and Rac1 GTPases, regulates cell protrusions and motility by controlling actin and adhesion dynamics. Its deregulation has been linked to human cancer. We show here that activation of PAK1 is necessary for protrusive activity during cell spreading. To investigate PAK1 activation dynamics at live protrusions, we developed a conformational biosensor, based on fluorescence resonance energy transfer. This novel PAK1 biosensor allowed the spatiotemporal visualization of PAK1 activation during spreading of COS-7 cells and during motility of normal rat kidney cells. By using this imaging approach in COS-7 cells, the following new insights on PAK1 regulation were unveiled. First, PAK1 acquires an intermediate semi-open conformational state upon recruitment to the plasma membrane. This semi-open PAK1 species is selectively autophosphorylated on serines in the N-terminal regulatory region but not on the critical threonine 423 in the catalytic site. Second, this intermediate PAK1 state is hypersensitive to stimulation by Cdc42 and Rac1. Third, interaction with PIX proteins contributes to PAK1 stimulation at membrane protrusions, in a GTPase-independent way. Finally, trans-phosphorylation events occur between PAK1 molecules at the membrane possibly playing a relevant role for its activation. This study leads to a model for the complex and accurate regulation of PAK1 kinase in vivo at cell protrusions.

Structures of Dimeric GIT1 and Trimeric β-PIX and Implications for GIT–PIX Complex Assembly

GIT (G protein-coupled receptor kinase-interacting protein) and PIX (p21-activated kinase-interacting exchange factor) family proteins integrate signaling pathways involving Arf and Rho family GTPases. GIT1 and beta-PIX form a constitutively associated complex that acts as a scaffold to allow the formation of large multiprotein assemblies that regulate synaptogenesis, cell polarity and cell migration among other physiological processes. Complex formation is mediated by the GIT binding domain (GBD) in beta-PIX, which recognizes the Spa homology domain of GIT1. Both binding domains are adjacent to predicted coiled-coil segments that allow homo-oligomerization of GIT1 and beta-PIX, respectively. Oligomerization of GIT and PIX proteins is important for their physiological functions, and deletion of the coiled-coil domains interferes with correct subcellular localization and the GEF (guanine nucleotide exchange factor) activity of PIX. We have solved the crystal structures of the CC domains of GIT1 and beta-PIX and determined the stoichiometry of complex formation between the two proteins in order to understand the molecular architecture of the GIT1-beta-PIX complex. The crystal structure of the CC domain of GIT1 solved at 1.4 A resolution shows a dimeric, parallel CC that spans 67 A in length. Unexpectedly, and in contrast to prevalent dimeric models, the structure of the CC region of beta-PIX determined at 2.8 A resolution, combined with hydrodynamic studies, reveals that this protein forms a parallel trimer. Furthermore, we demonstrate that dimeric GIT and trimeric PIX form an unusual high-affinity heteropentameric complex in which each Spa homology domain of the GIT1 dimer recognizes one GBD of the beta-PIX trimer, leaving one GBD unoccupied. These results can serve as a basis to better understand oligomerization-dependent GIT1-beta-PIX-regulated signaling events and provide an insight into the architecture of large signaling complexes involving GIT1 and beta-PIX.

Pix Proteins and the Evolution of Centrioles

We have made a wide phylogenetic survey of Pix proteins, which are constituents of vertebrate centrioles in most eukaryotes. We have also surveyed the presence and structure of flagella or cilia and centrioles in these organisms, as far as is possible from published information. We find that Pix proteins are present in a vast range of eukaryotes, but not all. Where centrioles are absent so are Pix proteins. If one considers the maintenance of Pix proteins over evolutionary time scales, our analysis would suggest that their key function is to make cilia and flagella, and the same is true of centrioles. Moreover, this survey raises the possibility that Pix proteins are only maintained to make cilia and flagella that undulate, and even then only when they are constructed by transporting ciliary constituents up the cilium using the intraflagellar transport (IFT) system. We also find that Pix proteins have become generally divergent within Ecdysozoa and between this group and other taxa. This correlates with a simplification of centrioles within Ecdysozoa and a loss or divergence of cilia/flagella. Thus Pix proteins act as a weathervane to indicate changes in centriole function, whose core activity is to make cilia and flagella.

αPIX Rho GTPase Guanine Nucleotide Exchange Factor Regulates Lymphocyte Functions and Antigen Receptor Signaling

AlphaPIX is a Rho GTPase guanine nucleotide exchange factor domain-containing signaling protein that associates with other proteins involved in cytoskeletal-membrane complexes. It has been shown that PIX proteins play roles in some immune cells, including neutrophils and T cells. In this study, we report the immune system phenotype of alphaPIX knockout mice. We extended alphaPIX expression experiments and found that whereas alphaPIX was specific to immune cells, its homolog betaPIX was expressed in a wider range of cells. Mice lacking alphaPIX had reduced numbers of mature lymphocytes and defective immune responses. Antigen receptor-directed proliferation of alphaPIX(-) T and B cells was also reduced, but basal migration was enhanced. Accompanying these defects, formation of T-cell-B-cell conjugates and recruitment of PAK and Lfa-1 integrin to the immune synapse were impaired in the absence of alphaPIX. Proximal antigen receptor signaling was largely unaffected, with the exception of reduced phosphorylation of PAK and expression of GIT2 in both T cells and B cells. These results reveal specific roles for alphaPIX in the immune system and suggest that redundancy with betaPIX precludes a more severe immune phenotype.

Pix1 and Pix2 are novel WD40 microtubule-associated proteins that colocalize with mitochondria in Xenopus germ plasm and centrosomes in human cells

In many animals, the germ line develops from a distinct mitochondria-rich region of embryonic cytoplasm called the germ plasm. However, the protein composition of germ plasm and its formation remain poorly understood, except in Drosophila. Here, we show that Xpat, a recently identified protein component of Xenopus germ plasm, interacts via its C-terminal domain with a novel protein, xPix1. Xpat and xPix1 are co-expressed in ovaries, eggs and early embryos and colocalize to the mitochondrial cloud and germ plasm in stage I and stage VI oocytes, respectively. Although Xpat appears unique to Xenopus, Pix proteins, which contain an N-terminal WD40 domain and C-terminal coiled-coil, are widely conserved. In humans, two proteins, Pix1 and Pix2, are expressed at varying levels in different cancer cell lines. Importantly, as well as localizing to mitochondria, human Pix proteins localize to centrosomes and associate with microtubules in vitro and in vivo. Although, Pix proteins are stably expressed through the cell cycle, Pix2 concentrates on microtubule structures in mitosis and microinjection of Pix antibodies interferes with cell division. Based on these data, we propose that Pix1 and Pix2 are microtubule-associated adaptor proteins that likely contribute to a range of developmental and cell division processes.

The GIT/PIX complex: an oligomeric assembly of GIT family ARF GTPase-activating proteins and PIX family Rac1/Cdc42 guanine nucleotide exchange factors

GIT proteins are GTPase-activating proteins (GAPs) for ADP-ribosylation factor (ARF) small GTP-binding proteins, and interact with the PIX family of Rac1/Cdc42 guanine nucleotide exchange factors. GIT and PIX transiently localize p21-activated protein kinases (PAKs) to remodeling focal adhesions through binding to paxillin. To understand the role of these interactions, the association of GIT and PIX proteins was examined in detail. Two separable binding interactions link GIT and PIX proteins, GIT and PIX proteins each dimerize and a β-PIX fragment containing the GIT-binding region failed to inhibit the association of the GIT and PIX proteins. Endogenous GIT and PIX co-fractionate at a very high molecular size. Purified 6xHis-tagged β-PIX from Sf9 cells co-expressing untagged GIT1 yields recombinant GIT1/β-PIX complexes that have equal amounts of β-PIX and GIT1 and co-fractionate at the same large size as native GIT/PIX complexes. Thus, GIT and PIX proteins are tightly associated as a multimeric nexus capable of linking together important signaling molecules, including PAKs.

134. Structural Requirements of Adenovirus Targeting Via by Modification of Minor Capsid Protein IX

In mastadenoviruses, the hexon-associated protein IX is a 14.3kDa protein that is strongly conserved between the different members of the genus. It associates with nine hexon capsomers to form group-of-nine hexons (GON). These make up the central region of each of the facets of the icosahedron. Protein IX is believed to function as a capsid 'cement'. So far, its structure in not known. Although viruses lacking pIX do not form GONs and are less heat-stable than wild-type viruses, they can be propagated with the same kinetics and yields as the wild-type viruses. Deletion mutants lacking (parts of) the conserved N-terminal domain are not incorporated into the virion, implicating this domain in capsid formation. In contrast, pIX molecules with mutations in other conserved domains (i.e. the central alanine stretch, and the putative leucine zipper domain) are efficiently incorporated into virions. The carboxyl terminal domain contains a putative leucine-zipper domain that may be responsible for trimerization via the formation of a coiled-coil structure. Mutants in this region are efficiently incorporated into the capsid, suggesting that trimerization is not essential for capsid formation. To get more insight into the structure/function relationship we studied the incorporation of heterologous pIX molecules into the HAdV-5 capsid. Our data show that the pIX proteins of HAdV-12 (species A) and HAdV-7 (species B) can be efficiently incorporated into the capsid of pIX-deleted HAdV-5 (species C). In addition, further deletion mutagenesis confirmed that the N-terminus of the HAdV-5 pIX is required and sufficient for incorporation into the capsid. These data further our knowledge on pIX and facilitated the development of pIX as an anchor for the incorporation of heterologous peptides into the capsid. In this way receptor-binding ligands were incorporated in the capsid. Inclusion of alphahelical spacers of up to 7.5 nm between the pIX anchor and the ligand resulted in more efficient binding of the virions to antibodies and cells. These data conform that modification of pIX can be used to incorporate heterologous peptides into the adenoviral capsid, and support the feasibility of this approach to amend the tissue tropism of the adenovirus vectors.

Sequence and transcription map analysis of early region-1 of porcine adenovirus type-3

The nucleotide sequence of a region of the genome of porcine adenovirus-3 (PAV-3) between map units 1 and 12.2 was determined. The sequenced region included four major open reading frames, and several transcription control elements. Homology studies, using the deduced amino acid sequences of the open reading frames, revealed genes coding for the E1A, E1B 202R, E1B 474R and pIX proteins. The region was characterized by Northern blot analysis and sequencing of cDNA clones. In PAV-3, the E1A region is located between 1.5 and 3.8 map units. Alternate splice donor sites are used to produce four different types of transcripts from the primary transcript of the E1A region. The E1A proteins of PAV-3 contain a consensus zinc finger motif, which was shown to be the principal transactivation region of human adenovirus-5 (HAV-5) E1A proteins. The PAV-3 E1A proteins also contain a retinoblastoma susceptibility protein (pRb) binding motif, which in HAVs interacts with cellular Rb protein to overcome the pRb mediated transcription repression. The E1B region in PAV-3 maps between 4.0 and 12.2 map units, and shares a polyadenylation signal and polyadenylation sites with the gene coding for pIX. A single major and a number of minor mRNA species are produced from the E1B region. The open reading frame (ORF) analysis of cDNA representing major mRNA produced from the E1B region showed two overlapping ORFs corresponding to 19K and 55K ORFs of HAV-2. In PAV-3, the gene coding for pIX is located between 9.9 and 12.2 map units and codes for a protein of 199 amino acids.

Development of Cell Lines Capable of Complementing E1, E4, and Protein IX Defective Adenovirus Type 5 Mutants

The cloning capacity of currently available E1- and E3-deleted adenovirus (Ad) vectors does not exceed 8 kb. To increase capacity and improve vector safety further, we have explored the possibility that Early Region 4 (E4) and the gene encoding protein IX (pIX) might also be deleted. To generate cell lines expressing sufficient levels of E4 and pIX proteins in trans in addition to E1-encoded proteins to complement mutations in these genes, we transformed 293 cells with constructs containing the E4 transcription unit and pIX coding sequences under the control of inducible mouse mammary tumor virus (MMTV) and metallothionein promoters, respectively. We obtained two lines, VK2-20 and VK10-9, that express both E4 and pIX proteins as well as E1. The lines could be efficiently transfected with DNA, and allowed the rescue and propagation of an adenovirus; recombinant, Ad5dlE3,4, containing a 2.7-kb E3 deletion and a 2.8-kb E4 deletion in addition to an insertion of plasmid DNA sequences in E1A. Because the E4 sequences within VK2-20 and VK10-9 cells do not overlap with the DNA sequence of Ad5dlE3,E4, the probability of regeneration of the wild-type E4 during virus propagation should be very low. Using the cell lines described in this study, it should be possible to generate Ad vectors lacking E1, pIX, E3, and E4. This would not only increase capacity over that of currently available vectors (to approximately 11 kb) but would also result in more severely attenuated vectors than those with deletions only of E1 or of E1 and E3 and, hence, safer for use in gene therapy protocols.