FF Domains Research Articles

Backbone 1H, 15N, and 13C resonance assignments of the FF1 domain from P190A RhoGAP in 5 and 8M urea.

The Rho GTPase (Ras homolog GTPases) system is a crucial signal transducer that regulates various cellular processes, including cell cycle and migration, genetic transcription, and apoptosis. In this study, we investigated the unfolded state of the first FF domain (FF1) of P190A RhoGAP, which features four tandem FF domains. For signal transduction, FF1 is phosphorylated at tyrosine 308 (Y308), which is buried in the hydrophobic core and is inaccessible to kinases in the folded domain. It was proposed, therefore, that the phosphorylation occurs in a transiently populated unfolded state of FF1. To probe the folding pathway of the RhoGAP FF1 domain, here we have performed a nearly complete backbone resonance assignments of a putative partially unfolded state of FF1 in 5M urea and its fully unfolded state in 8M urea.

HNRNPL Circularizes ARHGAP35 to Produce an Oncogenic Protein.

Circular RNAs (circRNAs) are an intriguing class of widely prevalent endogenous RNAs, the vast majority of which have not been characterized functionally. Here, we identified a novel oncogenic circRNA originating from the back‐splicing of Exon2 and Exon3 of a tumor suppressor gene, ARHGAP35 (also known as P190‐A), termed as circARHGAP35. have observe that circARHGAP35 and linear ARHGAP35 have antithetical expression and functions. Interestingly, circARHGAP35 contains a 3867 nt long ORF with an m6A‐modified start codon and encodes a truncated protein comprising four FF domains and lacking the Rho GAP domain. Mechanistically, circARHGAP35 protein promotes cancer cell progression by interacting with TFII‐I protein in the nucleus. The RNA binding protein, HNRNPL, facilitates the formation of circARHGAP35. Clinically, circARHGAP35 is associated with poor survival in cancer patients. Our findings characterize an oncogenic circRNA and demonstrate a novel mechanism of oncogene activation in cancer by circRNA through the production of a truncated protein.

Molecular biophysical characterization of the third FF domain of Homo sapiens Prp40 homolog A

Homo sapiens Prp40 Homolog A (HsPrp40A) is a nuclear protein that is involved in pre-mRNA splicing. This modular protein has two WW domains followed by six tandem FF domains. Previous studies demonstrate that the WW domain-mediated interactions of HsPrp40A are implicated in genetic disorders such as Huntington's disease and Rett syndrome. However, little is known about the biological implications related to HsPrp40A's FF domains. Recently, we determined that HsPrp40A peptide interacts with high affinity with a calcium-binding protein known as centrin using isothermal titration calorimetry and two-dimensional infrared correlation spectroscopy. Interestingly, this centrin-binding site is located within the third FF (FF3) domain of HsPrp40A. Herein, we present a comparative molecular biophysical study using circular dichroism and two-dimensional infrared correlation spectroscopy to explore the molecular behavior of HsPrp40A's FF3 domain under thermal stress. These results may provide new insights about the stability of HsPrp40A's FF3 domain.

Interaction of p190A RhoGAP with eIF3A and Other Translation Preinitiation Factors Suggests a Role in Protein Biosynthesis

The negative regulator of Rho family GTPases, p190A RhoGAP, is one of six mammalian proteins harboring so-called FF motifs. To explore the function of these and other p190A segments, we identified interacting proteins by tandem mass spectrometry. Here we report that endogenous human p190A, but not its 50% identical p190B paralog, associates with all 13 eIF3 subunits and several other translational preinitiation factors. The interaction involves the first FF motif of p190A and the winged helix/PCI domain of eIF3A, is enhanced by serum stimulation and reduced by phosphatase treatment. The p190A/eIF3A interaction is unaffected by mutating phosphorylated p190A-Tyr308, but disrupted by a S296A mutation, targeting the only other known phosphorylated residue in the first FF domain. The p190A-eIF3 complex is distinct from eIF3 complexes containing S6K1 or mammalian target of rapamycin (mTOR), and appears to represent an incomplete preinitiation complex lacking several subunits. Based on these findings we propose that p190A may affect protein translation by controlling the assembly of functional preinitiation complexes. Whether such a role helps to explain why, unique among the large family of RhoGAPs, p190A exhibits a significantly increased mutation rate in cancer remains to be determined.

Detect Fast-Flux Domains Through Response Time Differences

A fast-flux service network (FFSN) uses dynamic DNS to map a dynamic domain, called fast-flux domain (FF domain), to various IP addresses and uses flux bots to redirect network traffic. Due to its powerful capability to conceal the hosts hidden behind the flux bots, FFSNs are widely adopted by attackers to cover various scams. Although diverse promising solutions have been proposed to detect FF domains, they face the same problem-different countermeasures could be used to bypass their detection. Hence, it becomes a critical issue to develop a new detection solution. According to our survey, unlike normal network services that use dynamic DNS to balance the workloads of their hosts, FFSNs utilize dynamic DNS to hide important bots. As a result, the response time of subsequent requests to an FF domain becomes more fluctuating. Based on the response time differences, this paper develops a new metric, Fast-Flux Score (FF-Score), to detect FF domains. Our system, called fast-flux domain detector (FFDD), is used on a computer that could be an end host or an IDS. A user with a set of unknown URLs, which may be obtained from spam or social networks, can simply determine whether they are benign domains or fast-flux ones using FFDD. Experimental results show that FFDD can accurately detect FF domains with only a 0.3% false positive rate and a 2% false negative rate. It takes less than 20 min for FFDD to determine whether a domain is an FF domain. In addition, FFDD is a lightweight stand-alone system; hence, it does not require special support from an ISP or any other network service.

Visualizing Side Chains of Invisible Protein Conformers by Solution NMR

Sparsely populated and transiently formed protein conformers can play key roles in many biochemical processes. Understanding the structure function paradigm requires, therefore, an atomic-resolution description of these rare states. However, they are difficult to study because they cannot be observed using standard biophysical techniques. In the past decade, NMR methods have been developed for structural studies of these elusive conformers, focusing primarily on backbone 1H, 15N and 13C nuclei. Here we extend the methodology to include side chains by developing a 13C-based chemical exchange saturation transfer experiment for the assignment of side-chain aliphatic 13C chemical shifts in uniformly 13C labeled proteins. A pair of applications is provided, involving the folding of β-sheet Fyn SH3 and α-helical FF domains. Over 96% and 89% of the side-chain 13C chemical shifts for excited states corresponding to the unfolded conformation of the Fyn SH3 domain and a folding intermediate of the FF domain, respectively, have been obtained, providing insight into side-chain packing and dynamics.

Specific Interaction of the Transcription Elongation Regulator TCERG1 with RNA Polymerase II Requires Simultaneous Phosphorylation at Ser2, Ser5, and Ser7 within the Carboxyl-terminal Domain Repeat

The human transcription elongation regulator TCERG1 physically couples transcription elongation and splicing events by interacting with splicing factors through its N-terminal WW domains and the hyperphosphorylated C-terminal domain (CTD) of RNA polymerase II through its C-terminal FF domains. Here, we report biochemical and structural characterization of the C-terminal three FF domains (FF4-6) of TCERG1, revealing a rigid integral domain structure of the tandem FF repeat that interacts with the hyperphosphorylated CTD (PCTD). Although FF4 and FF5 adopt a classical FF domain fold containing three orthogonally packed α helices and a 310 helix, FF6 contains an additional insertion helix between α1 and α2. The formation of the integral tandem FF4-6 repeat is achieved by merging the last helix of the preceding FF domain and the first helix of the following FF domain and by direct interactions between neighboring FF domains. Using peptide column binding assays and NMR titrations, we show that binding of the FF4-6 tandem repeat to the PCTD requires simultaneous phosphorylation at Ser(2), Ser(5), and Ser(7) positions within two consecutive Y(1)S(2)P(3)T(4)S(5)P(6)S(7) heptad repeats. Such a sequence-specific PCTD recognition is achieved through CTD-docking sites on FF4 and FF5 of TCERG1 but not FF6. Our study presents the first example of a nuclear factor requiring all three phospho-Ser marks within the heptad repeat of the CTD for high affinity binding and provides a molecular interpretation for the biochemical connection between the Ser(7) phosphorylation enrichment in the CTD of the transcribing RNA polymerase II over introns and co-transcriptional splicing events.

Specific interaction of the TCERG1 FF4–6 tandem repeat domains with RNA polymerase II requires simultaneous phosphorylation at Ser2, Ser5 and Ser7 of the CTD

The human transcription elongation regulator TCERG1 physically couples transcription elongation and splicing events by interacting with splicing factors through its N‐terminal WW domains and the hyperphosphorylated C‐terminal domain (CTD) of RNA polymerase II (RNAPII) through its C‐terminal FF domains. Here, we report biochemical and structural characterization of the C‐terminal three FF domains (FF4–6) of TCERG1, revealing a rigid integral domain structure of the tandem FF domains that interacts with the hyperphosphorylated CTD (PCTD). Using peptide column binding assays and NMR titrations, we show that binding of FF4–6 toward the PCTD requires simultaneous phosphorylation at Ser2, Ser5 and Ser7 positions within the Y1S2P3T4S5P6S7 heptad repeats. Such a sequence‐specific PCTD recognition is achieved through CTD‐docking sites on FF4 and FF5 of TCERG1, but not FF6. In vivo yeast cell extract pull down assay demonstrates the essential role of Ser7P. Our study presents the first example of a nuclear factor requiring all three phosphoSer marks within the heptad repeat of the CTD for high‐affinity binding and provides a molecular interpretation for the biochemical connection between the Ser7P enrichment and co‐transcriptional splicing events. This research was supported by NIH grants GM079376 (to P.Z.) and GM040505 (A.L.G.).

Chemical synthesis of the third WW domain of TCERG 1 by native chemical ligation

The human transcription elongation regulator 1 (TCERG1), with its modular architecture of 3 WW and 6 FF domains, inhibits RNA polymerase II elongation through these WW and FF domains, and interacts with pre-mRNA splicing factors such as SF1, U2 snRNP and U2AF. WW domains are known as the smallest naturally occurring, monomeric, triple stranded, anti-parallel β-sheet structures, generally spanning only about 40 amino acids. The first and second WW domains of TCERG1 have been synthesized and successfully applied for screening cellular targets. In contrast, until now syntheses of the third WW domain yielded oligopeptides with an undefined fold, proving useless for screening cellular targets. This implied that sequence elongation to include the α-helical structure is crucial for proper folding. We describe here the chemical synthesis of such a 53 residue long TCERG1 WW-3 domain sequence that exhibits the typical WW domain fold, and could be useful for discovering cellular targets.

Differential Effects of Sumoylation on Transcription and Alternative Splicing by Transcription Elongation Regulator 1 (TCERG1)

Modification of proteins by small ubiquitin-like modifier (SUMO) is emerging as an important control of transcription and RNA processing. The human factor TCERG1 (also known as CA150) participates in transcriptional elongation and alternative splicing of pre-mRNAs. Here, we report that SUMO family proteins modify TCERG1. Furthermore, TCERG1 binds to the E2 SUMO-conjugating enzyme Ubc9. Two lysines (Lys-503 and Lys-608) of TCERG1 are the major sumoylation sites. Sumoylation does not affect localization of TCERG1 to the splicing factor-rich nuclear speckles or the alternative splicing function of TCERG1. However, mutation of the SUMO acceptor lysine residues enhanced TCERG1 transcriptional activity, indicating that SUMO modification negatively regulates TCERG1 transcriptional activity. These results reveal a regulatory role for sumoylation in controlling the activity of a transcription factor that modulates RNA polymerase II elongation and mRNA alternative processing, which are discriminated differently by this post-translational modification.

Solution structure of the fourth FF domain of yeast Prp40 splicing factor

Prp40 protein was originally identified as a suppressor of 50 end U1 RNA point mutations.1 Prp40 is a U1 snRNP-associated protein that participates in the early steps of yeast premessenger RNA splicing. Prp40 associates with the branch-binding point protein to bring the 50 splicing site and the intron branch point into spatial proximity.2 Additionally, Prp40 has been implicated in the binding to the phosphorylated C-terminal domain (herein referred as phospho-CTD) of RNA polymerase II through regions involving the WW and FF domains.3 However, a subsequent study on the structure of the Prp40 WW domain pair also showed that, in the absence of additional FF domains, the WW domains do not interact with the phospho-CTD repeats.4 The solution structure of the first FF domain of Prp40 has been determined.5 That study also examined the binding of Prp40FF1 to the splicing factor Clf1 and to a pair of bisphospho-CTD repeats. The binding site for the association with the first TPR motif of Clf1 involves the helices a2, the 310, and the N-terminal half of a3. In contrast, no interaction was detected for the Prp40FF1 domain with the phospho-CTD repeats and for the Prp40FF4 domain with the TPR motif of Clf1.5 Recently, other ligand partners have been identified for Prp40 FF domains, namely Snu71, a component of U1 snRNP, and Luc7, a splicing factor associated with U1 snRNP involved in the 50 splicing site recognition.6–8 Furthermore, it was shown that only the region comprising the first two FF domains of Prp40 is critical for yeast viability, whereas the deletion of the region including FF3 and FF4 results in a slow-growth phenotype.6 These results are in agreement with a previous report that showed that a deletion of the FF1 domain and a fragment of FF2 of Prp40 caused yeast lethality.9 To further study the remaining FF domains present in Prp40, we cloned and produced constructs corresponding to the FF2, FF3, and FF4 domains. Of these, only the constructs corresponding to FF4 gave a folded and stable sample. Indeed, several constructs spanning FF2 and FF3 and even that of the FF1-2 pair were either partially structured or unstable after refolding from inclusion bodies. Here we report the solution structure of the Prp40FF4 domain. Furthermore, prompted by the observation that the charge distribution of the FBP11FF1 region involved in the interaction with the bisphospho-CTD repeats10 is partially conserved in Prp40FF4, we also examined whether this domain also interacted with the phosphoCTD repeats, but no binding was detected under our experimental conditions.

Structural Studies of FF Domains of the Transcription Factor CA150 Provide Insights into the Organization of FF Domain Tandem Arrays

FF domains are poorly understood protein interaction modules that are present within eukaryotic transcription factors, such as CA150 (TCERG-1). The CA150 FF domains have been shown to mediate interactions with the phosphorylated C-terminal domain of RNA polymerase II (phosphoCTD) and a multitude of transcription factors and RNA processing proteins, and may therefore have a central role in organizing transcription. FF domains occur in tandem arrays of up to six domains, although it is not known whether they adopt higher-order structures. We have used the CA150 FF1 + FF2 domains as a model system to examine whether tandem FF domains form higher-order structures in solution using NMR spectroscopy. In the solution structure of FF1 fused to the linker that joins FF1 to FF2, we observed that the highly conserved linker peptide is ordered and forms a helical extension of helix α3, suggesting that the interdomain linker might have a role in orientating FF1 relative to FF2. However, examination of the FF1 + FF2 domains using relaxation NMR experiments revealed that although these domains are not rigidly orientated relative to one another, they do not tumble independently. Thus, the FF1 + FF2 structure conforms to a dumbbell-shape in solution, where the helical interdomain linker maintains distance between the two dynamic FF domains without cementing their relative orientations. This model for FF domain organization within tandem arrays suggests a general mechanism by which individual FF domains can manoeuvre to achieve optimal recognition of flexible binding partners, such as the intrinsically-disordered phosphoCTD.

Crystal Structure of the Three Tandem FF Domains of the Transcription Elongation Regulator CA150

FF domains are small protein–protein interaction modules that have two flanking conserved phenylalanine residues. They are present in proteins involved in transcription, RNA splicing, and signal transduction, and often exist in tandem arrays. Although several individual FF domain structures have been determined by NMR, the tandem nature of most FF domains has not been revealed. Here we report the 2.7-Å-resolution crystal structure of the first three FF domains of the human transcription elongation factor CA150. Each FF domain is composed of three α-helices and a 3 10 helix between α-helix 2 and α-helix 3. The most striking feature of the structure is that an FF domain is connected to the next by an α-helix that continues from helix 3 to helix 1 of the next. The consequent elongated arrangement allows exposure of many charged residues within the region that can be engaged in interaction with other molecules. Binding studies using a peptide ligand suggest that a specific conformation of the FF domains might be required to achieve higher-affinity binding. Additionally, we explore potential DNA binding of the FF construct used in this study. Overall, we provide the first crystal structure of an FF domain and insights into the tandem nature of the FF domains and suggest that, in addition to protein binding, FF domains might be involved in DNA binding.

PKCδ influences p190 phosphorylation and activity: Events independent of PKCδ-mediated regulation of endothelial cell stress fiber and focal adhesion formation and barrier function

Background We have shown that protein kinase Cδ (PKCδ) inhibition results in increased endothelial cell (EC) permeability and decreased RhoA activity; which correlated with diminished stress fibers (SF) and focal adhesions (FA). We have also shown co-precipitation of p190RhoGAP (p190) with PKCδ. Here, we investigated if PKCδ regulates p190 and whether PKCδ-mediated changes in SF and FA or permeability were dependent upon p190. Methods Protein–protein interaction and activity analyses were performed using co-precipitation assays. Analysis of p190 phosphorylation was performed using in vitro kinase assays. SF and FA were analyzed by immunofluorescence analyses. EC monolayer permeability was measured using electrical cell impedance sensor (ECIS) technique. Results Inhibition of PKCδ increased p190 activity, while PKCδ overexpression diminished p190 activity. PKCδ bound to and phosphorylated both p190FF and p190GTPase domains. p190 protein overexpression diminished SF and FA formation and RhoA activity. Disruption of SF and FA or increased permeability induced upon PKCδ inhibition, were not attenuated in EC in which the p190 isoforms were suppressed individually or concurrently. General significance Our findings suggest that while PKCδ can regulate p190 activity, possibly at the FF and/or GTPase domains, the effect of PKCδ inhibition on SF and FA and barrier dysfunction occurs through a pathway independent of p190.

NMR Structural Studies on Human p190-A RhoGAPFF1 Revealed that Domain Phosphorylation by the PDGF-Receptor α Requires Its Previous Unfolding

p190-A and -B Rho GAPs (guanosine triphosphatase activating proteins) are the only cytoplasmatic proteins containing FF domains. In p190-A Rho GAP, the region containing the FF domains has been implicated in binding to the transcription factor TFII-I. Moreover, phosphorylation of Tyr308 within the first FF domain inhibits this interaction. Because the structural determinants governing this mechanism remain unknown, we sought to solve the structure of the first FF domain of p190-A Rho GAP (RhoGAPFF1) and to study the potential impact of phosphorylation on the structure.We found that RhoGAPFF1 does not fold with the typical (α1–α2–310–α3) arrangement of other FF domains. Instead, the NMR data obtained at 285 K show an α1–α2–α3–α4 topology. In addition, we observed that specific contacts between residues in the first loop and the fourth helix are indispensable for the correct folding and stability of this domain.The structure also revealed that Tyr308 contributes to the domain hydrophobic core. Furthermore, the residues that compose the target motif of the platelet-derived growth factor receptor α kinase form part of the α3 helix. We observed that the phosphorylation reaction requires a previous step including domain unfolding, a process that occurs at 310 K. In the absence of phosphorylation, the temperature-dependent RhoGAPFF1 folding/unfolding process is reversible. However, phosphorylation causes an irreversible destabilization of the RhoGAPFF1 structure, which probably accounts for the inhibitory effect that it exerts on the TFII-I interaction.Our results link the ability of a protein domain to be phosphorylated with conformational changes in its three-dimensional structure.

Solution structure of the yeast URN1 splicing factor FF domain: Comparative analysis of charge distributions in FF domain structures—FFs and SURPs, two domains with a similar fold

FF domains are present in three protein families: the splicing factors formin binding protein 11 (FBP11), Prp40, and URN1, the transcription factor CA150, and the p190RhoGTPase-related proteins. This simplicity in distribution, however, is contrasted by the difficulty in defining their biological role. At best, the group of ligand FF domains can bind to form a motley crew with binding reports pointing also to negative/aromatic sequences, the tetratricopeptide repeat, the transcription factor TFII-I and even to RNA. To expand our knowledge on the FF domain, we selected the FF domain present in the URN1 yeast splicing factor as the subject for structural studies. The URN1 protein is one of the two known proteins containing only one FF domain, making it the most simplified representative of FF domain-containing splicing factors. The solution structure reveals that the domain adopts the classical FF fold, with a distinctive negatively charged patch on its surface. All available FF structures have a well-conserved fold but variable electrostatic patches on their surfaces. These patches are unconserved, even for domains with similar pK(a)s. To investigate potential binding sites in FF domains, we performed structural comparisons to other proteins with similar folds. In addition to the structures detected by SCOP, we included SURP domains, which also adopt the alpha1-alpha2-3(10)-alpha3 architecture. We observed that the main difference between all these structures resides in the orientation of the second helix. Remarkably, in DEK, SURP, and Prp40FF1 structures (the exception is the FBP11FF1 domain), the second helix participates in ligand recognition. Furthermore, SURP and Prp40FF1 binding sites also include the 3(10) helix, which forms a partially exposed hydrophobic cavity. This cavity is also present in at least CA150FF1 and FF2 structures. Thus, as with WW domains, the FF fold seems to have developed binding-site variations to accommodate an abundant and variable set of ligands.

The FF domains of yeast U1 snRNP protein Prp40 mediate interactions with Luc7 and Snu71

BackgroundThe FF domain is conserved across all eukaryotes and usually acts as an adaptor module in RNA metabolism and transcription. Saccharomyces cerevisiae encodes two FF domain proteins, Prp40, a component of the U1 snRNP, and Ypr152c, a protein of unknown function. The structure of Prp40, its relationship to other proteins within the U1 snRNP, and its precise function remain little understood.ResultsHere we have investigated the essentiality and interaction properties of the FF domains of yeast Prp40. We show that the C-terminal two FF domains of Prp40 are dispensable. Deletion of additional FF domains is lethal. The first FF domain of Prp40 binds to U1 protein Luc7 in yeast two-hybrid and GST pulldown experiments. FF domains 2 and 3 bind to Snu71, another known U1 protein. Peptide array screens identified binding sites for FF1-2 within Snu71 (NDVHY) and for FF1 within Luc7 (ϕ[FHL] × [KR] × [GHL] with ϕ being a hydrophobic amino acid).ConclusionPrp40, Luc7, and Snu71 appear to form a subcomplex within the yeast U1snRNP. Our data suggests that the N-terminal FF domains are critical for these interactions. Crystallization of Prp40, Luc7, and Snu71 have failed so far but co-crystallization of pairs or the whole tri-complex may facilitate crystallographic and further functional analysis.

Protein-Protein-Interaktionen der U1snRNP-Komponente Prp40 und deren FF-Domänen in Saccharomyces cerevisiae

The FF domain is conserved in all eukaryotes indicating an important biological role. Many FF domain proteins play a role in RNA metabolism and their FF domains typically occur in tandem arrays. The yeast splicing factor Prp40 an essential component of the U1snRNP contains four FF domains and appears to play a role in stabilization of the yeast commitment complex during spliceosome assembly. The function of the FF domains in this process is unclear. FF domains of Prp40 and the human proteins CA150 and HYPA/FBP11 are known to interact with the phosphorylated C-terminal domain (CTD) of the RNA polymerase II. By yeast genome-wide two-hybrid screens I found that FF domains of Prp40 specifically interact with Snu71 and Luc7, known components of the U1snRNP. Mapping experiments showed that the FF1 domain of Prp40 interacts with a C-terminal region within Luc7, also confirmed by in vitro binding assays, whereas FF2-3 domains interact with a C-terminal region within Snu71. Peptide array results showed an interaction region within Snu71 (NDVHY) with no similarity to the consensus motif for Luc7 (Φ[FHL]x(KR]x[GHL]) identified via substitution analysis. Mutations of involved amino acids within the Luc7 sequence showed not the expected effect in Y2H experiments with FF domains. Only a mutation of the lysine resulted in the loss of binding with Prp40, but only in slow growth of yeast cells with FF1. Also a peptide containing the identified interacting region from Luc7 did not block the binding of FF1 and Luc7 in competition assay. Comparison of the identified motif for Luc7 with previous characterised consensus sequences from FF domains revealed no similarity, indicating the identification of a novel binding site for FF-domains. To get more insight into the yeast U1snRNP interactions also all associated proteins were tested in Y2H experiments. But only the interaction from Prp40 with Snu71 and Luc7 was detected again, concluding that this complex is hold together also through protein-RNA interaction. In vivo FF-domain deletions in Prp40 produced a mutant with growth defect lacking FF domains four and three, probably due to a defect in splicing. This was tested by a functional splice test with two representative intron containing genes, with no influence on splicing. To achieve all intron containing genes in yeast also microarray-splice experiments were initiated to obtain genome wide results. Based on all interaction studies, we conclude, that these three U1snRNP proteins Prp40, Snu71 and Luc7 build a sub complex within the U1snRNP via FF-domain interactions, like U1-A, U1-70K and U1-C in the human U1snRNP.

Human transcription elongation factor CA150 localizes to splicing factor-rich nuclear speckles and assembles transcription and splicing components into complexes through its amino and carboxyl regions.

The human transcription elongation factor CA150 contains three N-terminal WW domains and six consecutive FF domains. WW and FF domains, versatile modules that mediate protein-protein interactions, are found in nuclear proteins involved in transcription and splicing. CA150 interacts with the splicing factor SF1 and with the phosphorylated C-terminal repeat domain (CTD) of RNA polymerase II (RNAPII) through its WW and FF domains, respectively. WW and FF domains may, therefore, serve to link transcription and splicing components and play a role in coupling transcription and splicing in vivo. In the study presented here, we investigated the subcellular localization and association of CA150 with factors involved in pre-mRNA transcriptional elongation and splicing. Endogenous CA150 colocalized with nuclear speckles, and this was not affected either by inhibition of cellular transcription or by RNAPII CTD phosphorylation. FF domains are essential for the colocalization to speckles, while WW domains are not required for colocalization. We also performed biochemical assays to understand the role of WW and FF domains in mediating the assembly of transcription and splicing components into higher-order complexes. Transcription and splicing components bound to a region in the amino-terminal part of CA150 that contains the three WW domains; however, we identified a region of the C-terminal FF domains that was also critical. Our results suggest that sequences located at both the amino and carboxyl regions of CA150 are required to assemble transcription/splicing complexes, which may be involved in the coupling of those processes.

P190 RhoGAP functions in tissue morphogenesis

The p190 family of Rho GTPase activating proteins (GAPs) includes two closely related proteins, p190A and p190B, that are potent inhibitors of Rho GTPase activity. They can each be regulated in response to the engagement of cell surface adhesion molecules, such as integrins, as well as by activated growth factor receptors, and they play essential roles in mouse embryogenesis. However, they appear to mediate distinct signaling pathways by virtue of non-overlapping sites of regulatory phosphorylation. For p190A, src kinases are important regulators of activity and p190A is the principal substrate of src kinase-mediated phosphorylation in the brain. In addition, growth factor receptors, such as the PDGF receptor, can directly phosphorylate p190A on one of its so-called FF domains. As a consequence, p190A dissociates from the transcription factor, TFII-I, which becomes free to translocate to the nucleus to activate gene transcription. p190B is regulated through distinct mechanisms. p190B can be directly phosphorylated on a single identified tyrosine residue by the activated insulin and IGF-1 receptors. This phosphorylation causes p190B to translocate to the lipid rafts of the plasma membrane, where it can potentially facilitate the inactivation of the Rho GTPase, which also becomes enriched within the lipid rafts. Thus, upon insulin/IGF-1 signaling, phosphorylation of p190B can promote Rho inactivation. Mechanisms by which p190B may function to link IGF-1 and integrin-mediated signals that influence Rho-dependent actin reorganization will be presented.