Proteomic identification of a PSF/p54nrb heterodimer as RNF43 oncoprotein-interacting proteins

RNF43 is an oncogenic RING finger protein overexpressed in colorectal cancer. To dissect its biological functions, we explored RNF43-interacting proteins by pull-down assay and MS. We identified a heterodimer, p54nrb and PSF, as RNF43's binding partners and confirmed their physical interaction in vivo by the co-immunoprecipitation experiment. Immunofluorescence analysis revealed that co-expression of PSF relocates RNF43 from the nuclear periphery to the nucleoplasm. Thus, proteomic identification of RNF43-associated proteins sheds light on its dynamic interaction network in nuclear events.     

p54nrb is a component of the snRNP-free U1A (SF-A) complex that promotes pre-mRNA cleavage during polyadenylation

The U1 snRNP-A (U1A) protein has been known for many years as a component of the U1 snRNP. We have previously described a form of U1A present in human cells in significant amounts that is not associated with the U1 snRNP or U1 RNA but instead is part of a novel complex of non-snRNP proteins that we have termed snRNP-free U1A, or SF-A. Antibodies that specifically recognize this complex inhibit in vitro splicing and polyadenylation of pre-mRNA, suggesting that this complex may play an important functional role in these mRNA-processing activities. This finding was underscored by the determination that one of the components of this complex is the polypyrimidine-tract-binding protein-associated splicing factor, PSF. In order to further our studies on this complex and to determine the rest of the components of the SF-A complex, we prepared several stable HeLa cell lines that overexpress a tandem-affinity-purification-tagged version of U1A (TAP-tagged U1A). Nuclear extract was prepared from one of these cell lines, line 107, and affinity purification was performed along with RNase treatment. We have used mass spectrometry analysis to identify the candidate factors that associate with U1A. We have now identified and characterized PSF, p54(nrb), and p68 as novel components of the SF-A complex. We have explored the function of this complex in RNA processing, specifically cleavage and polyadenylation, by performing immunodepletions followed by reconstitution experiments, and have found that p54(nrb) is critical.     

The putative Bacillus subtilis l,d-transpeptidase YciB is a lipoprotein that localizes to the cell poles in a divisome-dependent manner

Cell wall synthesis in bacteria is spatially organized by cytoskeletal structures. Common to all cell wall-bearing bacteria, the cytokinetic machinery localizes the cell wall synthesis to the site of septation. Recently, MinJ, a new component of the cytokinetic machinery, or divisome, of Bacillus subtilis has been described. MinJ is part of the division site selection system but also essential for correct assembly of the divisome. Here, I used the isolated PDZ domain of MinJ for co-elution experiments. One of the proteins that co-eluted was the so far uncharacterized, putative l,d-transpeptidase protein YciB. Evidence is shown that YciB localizes to the cell poles. YciB localization depends on the existence of a mature divisome, suggesting that l,d-transpeptidases are, like penicillin-binding proteins, part of the divisome.     

Myo1 localizes to phagosomes, some of which traffic to the nucleus in a Myo1-dependent manner in Tetrahymena thermophila

Myo1 is one of 13 myosins in Tetrahymena thermophila. Initially, twelve of the myosins in Tetrahymena were assigned to Class XX in the myosin superfamily but recently re-assigned to a subclass within Class XIV. In a previous study, we reported that genomic knockout of MYO1 affected phagocytosis and macronuclear amitosis. These two phenotypes have appeared disparate because a possible mechanism linking phagocytosis and amitosis was unknown. In the present study, Myo1 localization was investigated in order to further link machinery for phagocytosis and amitosis. Antibodies directed against the Myo1 motor domain detected an immunospecific polypeptide at 175-180 kDa on immunoblots of wild-type proteins. The 175-180 kDa polypeptide was not detected on immunoblots of proteins from the knockout strain. For immunofluorescence microscopy, cells were allowed to internalize fluorescent beads as markers for phagosomes. In wild-type cells, anti-Myo1 and anti-actin antibodies co-localized to the periphery of phagosomes and the macronucleus. In the MYO1-knockout strain only background fluorescence was observed with anti-Myo1 antibody. Confocal x-z series through macronuclei revealed fluorescent beads within the nucleoplasm. Statistical analysis showed a significant difference between the mean distributions of fluorescent beads in the nucleoplasm of wild-type and MYO1-knockout cells. A fluorescent dye was used to label plasma membrane in living cells. Dye-labeled vacuoles trafficked to the macronucleus. Trafficking of phagosomes to the macronucleus in a myosin-dependent manner is a novel finding and a possible mechanism for targeting myosin and actin to the nucleus.     

Identification of a neuronal laminin receptor: an Mr 200K/120K integrin heterodimer that binds laminin in a divalent cation-dependent manner

Neuronal interactions with extracellular matrix (ECM) components are crucial for axon growth and guidance during development and nerve regeneration. Laminin (LN), a prominent ECM glycoprotein, promotes neuronal survival and axon growth. To identify neuronal receptors for LN, we looked for cell surface proteins on the neuronal cell line B50 that bind LN. An integrin alpha/beta 1 dimeric receptor was identified and purified using lectin and LN affinity chromatography. The purified integrin contains two subunits with Mrs of 200 K and 120 K that bind LN specifically in the presence, but not the absence, of divalent cations (Ca2+/Mg2+ or Ca2+/Mn2+). The Mr 120 K protein was identified as the rat integrin beta 1 subunit using two beta 1 subunit-specific antibodies, and was shown to form a noncovalent complex with the Mr 200K putative alpha subunit. Since neurons and neuronal cell lines express similar integrin beta 1-class heterodimers that mediate attachment and process outgrowth on LN, the Mr 200K/120K complex identified here is likely to be an important laminin receptor used by neurons. This integrin may also mediate binding to LN by many nonneuronal cell types.     

Insulin forms amyloid in a strain-dependent manner: an FT-IR spectroscopic study

The presence of 20% (v/v) ethanol triggers growth of insulin amyloid with distinct infrared spectroscopic features, compared with the fibrils obtained under ambient conditions. Here we report that the two insulin amyloid types behave in the prion strain-like manner regarding seeding specificity and ability of the self-propagating conformational template to overrule unfavorable environmental factors and maintain the initial folding pattern. The type of the original seed has been shown to prevail over cosolvent effects and determines spectral position and width of the amide I' infrared band of the heterogeneously seeded amyloid. These findings imply that "strains" may be a common generic trait of amyloids.     

ATP9B, a P4-ATPase (a putative aminophospholipid translocase), localizes to the trans-Golgi network in a CDC50 protein-independent manner

Type IV P-type ATPases (P4-ATPases) are putative phospholipid flippases that translocate phospholipids from the exoplasmic (lumenal) to the cytoplasmic leaflet of lipid bilayers and are believed to function in complex with CDC50 proteins. In Saccharomyces cerevisiae, five P4-ATPases are localized to specific cellular compartments and are required for vesicle-mediated protein transport from these compartments, suggesting a role for phospholipid translocation in vesicular transport. The human genome encodes 14 P4-ATPases and three CDC50 proteins. However, the subcellular localization of human P4-ATPases and their interactions with CDC50 proteins are poorly understood. Here, we show that class 5 (ATP10A, ATP10B, and ATP10D) and class 6 (ATP11A, ATP11B, and ATP11C) P4-ATPases require CDC50 proteins, primarily CDC50A, for their exit from the endoplasmic reticulum (ER) and final subcellular localization. In contrast, class 2 P4-ATPases (ATP9A and ATP9B) are able to exit the ER in the absence of exogenous CDC50 expression: ATP9B, but not ATP11B, was able to exit the ER despite depletion of CDC50 proteins by RNAi. Although ATP9A and ATP9B show a high overall sequence similarity, ATP9A localizes to endosomes and the trans-Golgi network (TGN), whereas ATP9B localizes exclusively to the TGN. A chimeric ATP9 protein in which the N-terminal cytoplasmic region of ATP9A was replaced with the corresponding region of ATP9B was localized exclusively to the Golgi. These results indicate that ATP9B is able to exit the ER and localize to the TGN independently of CDC50 proteins and that this protein contains a Golgi localization signal in its N-terminal cytoplasmic region.     

Cardiac phospholipase D2 localizes to sarcolemmal membranes and is inhibited by alpha-actinin in an ADP-ribosylation factor-reversible manner

Myocardial phospholipase D (PLD) has been implicated in the regulation of Ca(2+) mobilization and contractile performance in the heart. However, the molecular identity of this myocardial PLD and the mechanisms that regulate it are not well understood. Using subcellular fractionation and Western blot analysis, we found that PLD2 is the major myocardial PLD and that it localizes primarily to sarcolemmal membranes. A 100-kDa PLD2-interacting cardiac protein was detected using a protein overlay assay employing purified PLD2 and then identified as alpha-actinin using peptide-mass fingerprinting with matrix-assisted laser desorption/ionization mass spectroscopy. The direct association between PLD2 and alpha-actinin was confirmed using an in vitro binding assay and localized to PLD2's N-terminal 185 amino acids. Purified alpha-actinin potently inhibits PLD2 activity (IC(50) = 80 nm) in an interaction-dependent and ADP-ribosylation factor-reversible manner. Finally, alpha-actinin co-localizes with actin and with PLD2 in the detergent-insoluble fraction from sarcolemmal membranes. These results suggest that PLD2 is reciprocally regulated in sarcolemmal membranes by alpha-actinin and ARF1 and accordingly that a major role for PLD2 in cardiac function may involve reorganization of the actin cytoskeleton.     

AFAP1L1 is a novel adaptor protein of the AFAP family that interacts with cortactin and localizes to invadosomes

The actin-filament associated protein (AFAP) family of adaptor proteins consists of three members: AFAP1, AFAP1L1, and AFAP1L2/XB130 with AFAP1 being the best described as a cSrc binding partner and actin cross-linking protein. A homology search of AFAP1 recently identified AFAP1L1 which has a similar sequence, domain structure and cellular localization; however, based upon sequence variations, AFAP1L1 is hypothesized to have unique functions that are distinct from AFAP1. While AFAP1 has the ability to bind to the SH3 domain of the nonreceptor tyrosine kinase cSrc via an N-terminal SH3 binding motif, it was unable to bind cortactin. However, the SH3 binding motif of AFAP1L1 was more efficient at interacting with the SH3 domain of cortactin and not cSrc. AFAP1L1 was shown by fluorescence microscopy to decorate actin filaments and move to punctate actin structures and colocalize with cortactin, consistent with localization to invadosomes. Upon overexpression in A7r5 cells, AFAP1L1 had the ability to induce podosome formation and move to podosomes without stimulation. Immunohistochemical analysis of AFAP1L1 in human tissues shows differential expression when contrasted with AFAP1 with localization of AFAP1L1 to unique sites in muscle and the dentate nucleus of the brain where AFAP1 was not detectable. We hypothesize AFAP1L1 may play a similar role to AFAP1 in affecting changes in actin filaments and bridging interactions with binding partners, but we hypothesize that AFAP1L1 may forge unique protein interactions in which AFAP1 is less efficient, and these interactions may allow AFAP1L1 to affect invadosome formation.     

Scp160p, an RNA-binding, polysome-associated protein, localizes to the endoplasmic reticulum of Saccharomyces cerevisiae in a microtubule-dependent manner

Scp160p is an RNA-binding protein containing 14 tandemly repeated heterogenous nuclear ribonucleoprotein K-homology domains, which are implicated in RNA binding. Scp160p interacts with free and membrane-bound polysomes that are dependent upon the presence of mRNA. Despite its presence on cytosolic polysomes, Scp160p is predominantly localized to the endoplasmic reticulum (ER). Accumulation of Scp160p-ribosome complexes at the ER requires the function of microtubules but is independent of the actin cytoskeleton. We propose that the multi-K-homology-domain protein Scp160p functions as an RNA binding platform, interacting with polysomes that are transported to the ER.     

GIT1 is a novel MEK1–ERK1/2 scaffold that localizes to focal adhesions

Cell polarity is critical for cell migration and requires localized signal transduction in subcellular domains. Recent evidence demonstrates that activation of ERK1/2 (extracellular‐signal‐regulated kinase 1/2) in focal adhesions is essential for cell migration. GIT1 (G‐protein‐coupled receptor kinase‐interacting protein 1) has been shown to bind paxillin and regulate focal‐adhesion disassembly. We have previously reported that GIT1 binds to MEK1 [MAPK (mitogen‐activated protein kinase)/ERK kinase 1] and acts as a scaffold to enhance ERK1/2 activation in response to EGF (epidermal growth factor). In the present study we show that GIT1 associates with ERK1/2 in focal adhesions and this association increases after EGF stimulation. The CC (coiled‐coil) domain of ERK1/2 is required for association with GIT1, translocation to focal adhesions, and cell spreading and migration. Immunofluorescent staining showed that, after EGF stimulation, GIT1 co‐localized with pERK1/2 (phosphorylated ERK1/2) in focal adhesions. The binding of GIT1 and ERK1/2 was functionally important, since transfecting an ERK2 mutant lacking the CC domain [ERK2(del CC)] significantly decreased pERK1/2 translocation to focal adhesions, cell spreading and migration induced by EGF. In summary, the CC domain of ERK1/2 is necessary for binding to GIT1, for ERK1/2 activation in focal adhesions, and for cell spreading and migration.     

Impaired DNA damage response in cells expressing an exon 11-deleted murine Brca1 variant that localizes to nuclear foci

Both human and mouse cells express an alternatively spliced variant of BRCA1, BRCA1-Delta11, which lacks exon 11 in its entirety, including putative nuclear localization signals. Consistent with this, BRCA1-Delta11 has been reported to reside in the cytoplasm, a localization that would ostensibly preclude it from playing a role in the nuclear processes in which its full-length counterpart has been implicated. Nevertheless, the finding that murine embryos bearing homozygous deletions of exon 11 survive longer than embryos that are homozygous for Brca1 null alleles suggests that exon 11-deleted isoforms may perform at least some of the functions of Brca1. We have analyzed both the full-length and the exon 11-deleted isoforms of the murine Brca1 protein. Our results demonstrate that full-length murine Brca1 is identical to human BRCA1 with respect to its cell cycle regulation, DNA damage-induced phosphorylation, nuclear localization, and association with Rad51. Surprisingly, we show that endogenous Brca1-Delta11 localizes to discrete nuclear foci indistinguishable from those found in wild-type cells, despite the fact that Brca1-Delta11 lacks previously defined nuclear localization signals. However, we further show that DNA damage-induced phosphorylation of Brca1-Delta11 is significantly reduced compared to full-length Brca1, and that gamma irradiation-induced Rad51 focus formation is impaired in cells in which only Brca1-Delta11 is expressed. Our results suggest that the increased viability of embryos bearing homozygous deletions of exon 11 may be due to expression of Brca1-Delta11 and suggest an explanation for the genomic instability that accompanies the loss of full-length Brca1.     

Peflin and ALG-2, members of the penta-EF-hand protein family, form a heterodimer that dissociates in a Ca2+-dependent manner

Peflin, a newly identified 30-kDa Ca(2+)-binding protein, belongs to the penta-EF-hand (PEF) protein family, which includes the calpain small subunit, sorcin, grancalcin, and ALG-2 (apoptosis-linked gene 2). We prepared a monoclonal antibody against human peflin. The antibody immunoprecipitated a 22-kDa protein as well as the 30-kDa protein from the lysate of Jurkat cells. Western blotting of the immunoprecipitates revealed that the 22-kDa protein corresponds to ALG-2. This was confirmed by Western blotting of the immunoprecipitates of epitope-tagged peflin or ALG-2 whose cDNA expression constructs were transfected to human embryonic kidney (HEK) 293 cells. Gel filtration of the cytosolic fraction of Jurkat cells revealed co-elution of peflin and ALG-2 in fractions eluting earlier than recombinant ALG-2, further supporting the notion of heterodimerization of the two PEF proteins. Surprisingly, peflin dissociated from ALG-2 in the presence of Ca(2+). Peflin and ALG-2 co-localized in the cytoplasm, but ALG-2 was also detected in the nuclei as revealed by immunofluorescent staining and subcellular fractionation. Peflin was recovered in the cytosolic fraction in the absence of Ca(2+) but in the membrane/cytoskeletal fraction in the presence of Ca(2+). These results suggest that peflin has features common to those of other PEF proteins (dimerization and translocation to membranes) and may modulate the function of ALG-2 in Ca(2+) signaling.     

S1P1 localizes to the colonic vasculature in ulcerative colitis and maintains blood vessel integrity

Signaling through sphingosine-1-phosphate receptor₁ (S1P₁) promotes blood vessel barrier function. Degradation of S1P₁ results in increased vascular permeability in the lung and may explain side effects associated with administration of FTY720, a functional antagonist of the S1P₁ receptor that is currently used to treat multiple sclerosis. Ulcerative colitis (UC) is characterized by an increased density of abnormal vessels. The expression or role of S1P₁ in blood vessels in the colon has not been investigated. In the present study, we show that S1P₁ is overexpressed in the colonic mucosa of UC patients. This increase in S1P₁ levels reflects increased vascular density in the inflamed mucosa. Genetic deletion of S1pr1 in mice increases colonic vascular permeability under basal conditions and increases bleeding in experimental colitis. In contrast, neither FTY720 nor AUY954, two S1P receptor-targeting agents, increases bleeding in experimental colitis. Taken together, our findings demonstrate that S1P₁ is critical to maintaining colonic vascular integrity and may play a role in UC pathogenesis.