Polyproline Stretches Research Articles

EF-P Is Essential for Rapid Synthesis of Proteins Containing Consecutive Proline Residues

Elongation factor P (EF-P) is a translation factor of unknown function that has been implicated in a great variety of cellular processes. Here, we show that EF-P prevents ribosome from stalling during synthesis of proteins containing consecutive prolines, such as PPG, PPP, or longer proline strings, in natural and engineered model proteins. EF-P promotes peptide-bond formation and stabilizes the peptidyl-transfer RNA in the catalytic center of the ribosome. EF-P is posttranslationally modified by a hydroxylated β-lysine attached to a lysine residue. The modification enhances the catalytic proficiency of the factor mainly by increasing its affinity to the ribosome. We propose that EF-P and its eukaryotic homolog, eIF5A, are essential for the synthesis of a subset of proteins containing proline stretches in all cells.

Translation Elongation Factor EF-P Alleviates Ribosome Stalling at Polyproline Stretches

Translation elongation factor P (EF-P) is critical for virulence in bacteria. EF-P is present in all bacteria and orthologous to archaeal and eukaryotic initiation factor 5A, yet the biological function has so far remained enigmatic. Here, we demonstrate that EF-P is an elongation factor that enhances translation of polyproline-containing proteins: In the absence of EF-P, ribosomes stall at polyproline stretches, whereas the presence of EF-P alleviates the translational stalling. Moreover, we demonstrate the physiological relevance of EF-P to fine-tune the expression of the polyproline-containing pH receptor CadC to levels necessary for an appropriate stress response. Bacterial, archaeal, and eukaryotic cells have hundreds to thousands of polyproline-containing proteins of diverse function, suggesting that EF-P and a/eIF-5A are critical for copy-number adjustment of multiple pathways across all kingdoms of life.

Molecular cloning and expression analysis of stress-inducible protein I gene of Miichthys miiuy.

The full length of stress-inducible protein Ⅰ(STI1) gene cDNA was cloned from Miichthys miiuy based on the obtained EST sequence using rapid amplification of cDNA ends polymerase chain reaction(RACE-PCR).The STI1 gene cDNA consisted of 2 194 nucleotides including 5' untranslated regions(UTR) of 31 nucleotides,3' UTR of 634 nucleotides and open reading frame with 1 629 nucleotides encoding 542 amino acid residues.The molecular structure of STI1 consisted of five potential casein kinase Ⅱ phosphorylation sites,one polyproline stretch,two nuclear localization signals and nine tetratricopeptide repeats(TPR).The amino acid sequence of the STI1 gene shared 76.5%-94.3% identity with those of previously reported other species STI1 gene including conserved eight cysteines.Phylogenetic tree analysis based on amino acid sequences of STI1 gene generated by neighbor-joining method suggested that four teleosts clustered together,and were close genetic relationship between M.miiuy and Pseudosciaena crocea.Real-time PCR showed that the STI1 gene was mainly detected in muscle and kidney.

Implications Of Cis Interactions Between Expanded Polyglutamine and the Proline Rich C-Terminal Domain of Huntingtin Exon 1 For the Loss- Versus Gain-Of-Function Models of Huntington's Disease

At least nine neurodegenerative disorders, including Huntington's disease (HD), have been associated with expanded polyglutamine (polyQ) tracts. Proteolytic products of these proteins form neuronal intranuclear inclusions. There is growing interest in the role of naturally occurring flanking sequences and persistent controversy regarding their role as gatekeepers against or enhancers of polyQ aggregation. In particular, previous studies have shown that the 17-residue N-terminal segment (N17) of huntingtin (htt) exon 1 acts as gatekeepers, although controversy lingers with regards to this finding. Here, we focus on the interactions in cis between polyQ and the proline rich C-terminal domain (C38) of htt exon 1. This domain possesses two polyproline stretches (polyP) that appear to interact with profilin, which inhibits htt aggregation in cell culture studies.Through the use of atomistic Monte Carlo simulations utilizing the ABSINTH implicit solvation model, we show evidence for long-range interactions between polyQ tracts and both polyP regions. The polyQ segments promote the formation of cis peptide bonds within the polyP segment directly C-terminal to polyQ. This in turn will have important changes to the heterotypic interactions of htt through its polyP regions. A detailed comparative analysis of the monomer ensembles of N17-polyQ, polyQ-C38 and N17-polyQ-C38 constructs will be presented and the implications of our findings for loss- versus gain-of-function models for the onset and progression of disease will be discussed.This work was supported by grant 5R01NS056114 from the National Institutes of Health

Insights into the molecular regulation of FasL (CD178) biology

Fas ligand (FasL, CD95L, APO-1L, CD178, TNFSF6, APT1LG1) is the key death factor of receptor-triggered programmed cell death in immune cells. FasL/Fas-dependent apoptosis plays a pivotal role in activation-induced cell death, termination of immune responses, elimination of autoreactive cells, cytotoxic effector function of T and NK cells, and the establishment of immune privilege. Deregulation or functional impairment of FasL threatens the maintenance of immune homeostasis and defense and results in severe autoimmunity. In addition, FasL has been implicated as an accessory or costimulatory receptor in T cell activation. The molecular mechanisms underlying this reverse signaling capacity are, however, poorly understood and still controversially discussed. Many aspects of FasL biology have been ascribed to selective protein–protein interactions mediated by a unique polyproline region located in the membrane-proximal intracellular part of FasL. Over the past decade, we and others identified a large number of putative FasL-interacting molecules that bind to this polyproline stretch via Src homology 3 or WW domains. Individual interactions were analyzed in more detail and turned out to be crucial for the lysosomal storage, the transport and the surface appearance of the death factor and potentially also for reverse signaling. This review summarizes the work in the framework of the Collaborative Research Consortium 415 (CRC 415) and provides facts and hypotheses about FasL-interacting proteins and their potential role in FasL biology.

Minicollagen-15, a Novel Minicollagen Isolated from Hydra, Forms Tubule Structures in Nematocysts

Minicollagens constitute a family of unusually short collagen molecules isolated from cnidarians. They are restricted to the nematocyst, a cylindrical explosive organelle serving in defense and capture of prey. The nematocyst capsule contains a long tubule inside of its matrix, which is expelled and everted during an ultrafast discharge process. Here, we report the cloning and characterization of a novel minicollagen in Hydra, designated minicollagen-15 (NCol-15). NCol-15, like NCol-3 and NCol-4, shows deviations from the canonical cysteine pattern in its terminal cysteine-rich domains (CRDs). Minicollagens share common domain architectures with a central collagen sequence flanked by polyproline stretches and short N- and C-terminal CRDs. The CRDs are involved in the formation of a highly resistant cysteine network, which constitutes the basic structure of the nematocyst capsule. Unlike NCol-1, which is part of the capsule wall, NCol-15 is localized to tubules, arguing for a functional differentiation of minicollagens within the nematocyst architecture. NMR analysis of the altered C-terminal CRD of NCol-15 showed a novel disulfide-linked structure within the cysteine-containing region exhibiting similar folding kinetics and stability as the canonical CRDs. Our data provide evidence for evolutionary diversification among minicollagens, which probably facilitated alterations in the morphology of the nematocyst wall and tubule.

Mammalian Gene PEG10 Expresses Two Reading Frames by High Efficiency –1 Frameshifting in Embryonic-associated Tissues

Paternally expressed gene 10 (PEG10) is a mammalian gene that is essential for embryonic development in mice. The gene contains two overlapping open reading frames (ORF1 and ORF2) and is derived from a retroelement that acquired a cellular function. It is not known if both reading frames are required for PEG10 function. Synthesis of ORF2 would be possible only if programmed -1 frameshifting occurred during ORF1 translation. In this study the frameshifting activity of PEG10 was analyzed in vivo, and a potential role for ORF2 was investigated. Phylogenetic analysis demonstrated that PEG10 is highly conserved in therian mammals, with all species retaining the elements necessary for frameshifting as well as functional motifs in each ORF. The frameshift site of PEG10 was highly active in cultured cells and produced the ORF1-2 protein. In mice, endogenous ORF1 and an ORF1-2 frameshift protein were detected in the developing placenta and amniotic membrane from 9.5 days post-coitus through to term with a very high frameshift efficiency (>60%). Mutagenesis of the active site motif of a putative protease within ORF2 showed that this enzyme is active and participates in post-translational processing of PEG10 ORF1-2. Both PEG10 proteins were also detected in first trimester human placenta. By contrast, neither protein expression nor frameshifting was detected in adult mouse tissues. These studies imply that the ORF1-2 protein, synthesized utilizing the most efficient -1 frameshift mechanism yet documented in vivo, will have an essential function that is intrinsic to the importance of PEG10 in mammals.

Assembly of Acetylcholinesterase Tetramers by Peptidic Motifs from the Proline-rich Membrane Anchor, PRiMA: COMPETITION BETWEEN DEGRADATION AND SECRETION PATHWAYS OF HETEROMERIC COMPLEXES

The membrane-bound form of acetylcholinesterase (AChE) constitutes the major component of this enzyme in the mammalian brain. These molecules are hetero-oligomers, composed of four AChE catalytic subunits of type T (AChE(T)), associated with a transmembrane protein of type 1, called PRiMA (proline-rich membrane anchor). PRiMA consists of a signal peptide, an extracellular domain that contains a proline-rich motif (14 prolines with an intervening leucine, P4LP10), a transmembrane domain, and a cytoplasmic domain. Expression of AChE(T) subunits in transfected COS cells with a truncated PRiMA, without its transmembrane and cytoplasmic domains (P(stp54) mutant), produced secreted heteromeric complexes (T4-P(stp54)), instead of membrane-bound tetramers. In this study, we used a series of deletions and point mutations to analyze the interaction between the extracellular domain of PRiMA and AChE(T) subunits. We confirmed the importance of the polyproline stretches and defined a peptidic motif (RP4LP10RL), which induces the assembly and secretion of a heteromeric complex with four AChE(T) subunits, nearly as efficiently as the entire extracellular domain of PRiMA. It is noteworthy that deletion of the N-terminal segment preceding the prolines had little effect. Interestingly, short PRiMA mutants, truncated within the proline-rich motif, reduced both cellular and secreted AChE activity, suggesting that their interaction with AChE(T) subunits induces their intracellular degradation.

Tumor Cell-Derived HLA-B-Associated Transkript 3 (BAT3) Is a Ligand for NKp30 and Activates NK Cells.

Natural-killer (NK)-cells are lymphocytes of the innate immune system, which directly attack tumor and virus-infected cells. They provide a link between innate and adaptive immunity through crosstalk with dendritic cells (DCs) and mediate T-cell activation. Among the activating and inhibitory receptors that regulate NK cell activity, the orphan NKp30 receptor (NCR3, CD337) plays a special role as NKp30 does not only induce target cell lysis but is also crucial for the interaction between NK cells and dendritic cells. However, so far the cellular ligands for NKp30 have remained elusive.Using a yeast two hybrid approach with the extracellular NKp30 sequence as bait we were able to isolate a putative NKp30 ligand from a tumor cDNA library. Sequence analysis revealed that the cDNA encoded for BAT3 (HLA-B-associated transcript 3) which originally had been cloned from the human major histocompatibility complex by chromosome walking and mapped to chromosome 6p21.3 within the HLA complex. BAT3 was described as a nuclear protein characterized by an N-terminal ubiquitin-like region, a polyproline stretch and a highly conserved BAG-(Bcl-2-associated athanogene) domain. Until now, the function of BAT3 in mammals is not clearly defined. With BAT3-specific antibodies and by means of laser scanner microscopy and Western Blotting we demonstrate that tumor cell lines and immature as well as mature DCs express BAT3. Upon co-cultivation with NK cells or exposure to stress signals such as heat shock, we observed that BAT3 translocates from the nucleus to the cell-surface and the protein is released from tumor cells into the extracellular environment. BAT3 binds directly and specifically to NKp30 on NK cells and triggers NK cell-mediated cytokine release and NKp30-dependent cytotoxicity. The inhibition of endogenous BAT3 using polyclonal antibodies prevents tumor rejection in vivo, demonstrating that BAT3 is necessary for the tumor rejection in a xenograft tumor model.Thus, BAT3 is the long sought cellular NKp30 ligand and exhibits a novel mechanism by which a nuclear protein activates NK cells via NKp30-engagement after its release to the cell-surface and the cell environment. The isolation of the first cellular NKp30-ligand provides the basis for a better molecular understanding of NK cell-regulation and allows the development of clinical applications targeting NKp30 for the immunotherapy.

Identification of endophilins 1 and 3 as selective binding partners for VGLUT1 and their co-localization in neocortical glutamatergic synapses: implications for vesicular glutamate transporter trafficking and excitatory vesicle formation.

1. Selective protein-protein interactions between neurotransmitter transporters and their synaptic targets play important roles in regulating chemical neurotransmission. We screened a yeast two-hybrid library with bait containing the C-terminal amino acids of VGLUT1 and obtained clones that encode endophilin 1 and endophilin 3, proteins considered to play an integral role in glutamatergic vesicle formation. 2. Using a modified yeast plasmid vector to enable more cost-effective screens, we analyzed the selectivity and specificity of this interaction. Endophilins 1 and 3 selectively recognize only VGLUT1 as the C-terminus of VGLUT2 and VGLUT3 do not interact with either endophilin isoform. We mutagenized four conserved stretches of primary sequence in VGLUT1 that includes two polyproline motifs (Pro1, PPAPPP, and Pro2, PPRPPPP), found only in VGLUT1, and two conserved stretches (SEEK, SYGAT), found also in VGLUT2 and VGLUT3. The absence of the VGLUT conserved regions does not affect VGLUT1-endophilin association. Of the two polyproline stretches, only one (Pro2) is required for binding specificity to both endophilin 1 and endophilin 3. 3. We also show that endophilin 1 and endophilin 3 co-localize with VGLUT1 in synaptic terminals of differentiated rat neocortical neurons in primary culture. These results indicate that VGLUT1 and both endophilins are enriched in a class of excitatory synaptic terminals in cortical neurons and there, may interact to play an important role affecting the vesicular sequestration and synaptic release of glutamate.

A novel Dictyostelium RasGEF required for chemotaxis and development

BackgroundRas proteins are guanine-nucleotide-binding enzymes that couple cell surface receptors to intracellular signaling pathways controlling cell proliferation and differentiation, both in lower and higher eukaryotes. They act as molecular switches by cycling between active GTP and inactive GDP-bound states, through the action of two classes of regulatory proteins: a) guanine nucleotide exchange factor (GEFs) and b) GTP-ase activating proteins (GAPs). Genome wide analysis of the lower eukaryote Dictyostelium discoideum revealed a surprisingly large number of Ras Guanine Nucleotide Exchange Factors (RasGEFs). RasGEFs promote the activation of Ras proteins by catalyzing the exchange of GDP for GTP, thus conferring to RasGEFs the role of main activator of Ras proteins. Up to date only four RasGEFs, which are all non-redundant either for growth or development, have been characterized in Dictyostelium. We report here the identification and characterization of a fifth non-redundant GEF, RasGEFM.ResultsRasGEFM is a multi-domain protein containing six poly-proline stretches, a DEP, RasGEFN and RasGEF catalytic domain. The rasGEFM gene is differentially expressed during growth and development. Inactivation of the gene results in cells that form small, flat aggregates and fail to develop further. Expression of genes required for aggregation is delayed. Chemotaxis towards cAMP is impaired in the mutant, due to inability to inhibit lateral pseudopods. Endogenous cAMP accumulates during early development to a much lower extent than in wild type cells. Adenylyl cyclase activation in response to cAMP pulses is strongly reduced, by contrast guanylyl cyclase is stimulated to higher levels than in the wild type. The actin polymerization response to cAMP is also altered in the mutant. Cyclic AMP pulsing for several hours partially rescues the mutant. In vitro experiments suggest that RasGEFM acts downstream of the cAMP receptor but upstream of the G protein.ConclusionThe data indicate that RasGEFM is involved in the establishment of the cAMP relay system. We propose that RasGEFM is a component of a Ras regulated pathway, which integrate signals acting as positive regulator for adenylyl cyclase and negative regulator for guanylyl cyclase. Altered guanylyl cyclase, combined with defective regulation of actin polymerization, results in altered chemotaxis.

Genes Specifically Expressed in Sexually Differentiated Female Spheroids of Volvox carteri

Volvox carteri is a multicellular green alga with only two cell types, somatic cells and reproductive cells. Phylogenetic analysis suggests that this organism has evolved from a Chlamydomonas-like unicellular ancestor along with multicellularity, cellular differentiation, and a change in the mode of sexual reproduction from isogamy to oogamy. To examine the mechanism of sexual differentiation and the evolution of oogamy, we isolated 6 different cDNA sequences specifically expressed in sexually differentiated female spheroids. The genes for the cDNAs were designated SEF1 to SEF6. The time course of accumulation of each mRNA was shown to be distinct. The expression of some of these genes was not significantly affected when the sexual inducer was removed after the induction of sexual development. Sequence analysis indicates that SEF5 and SEF6 encode pherophorin-related proteins. Of these, SEF5 has the unique structural feature of a polyproline stretch in the C-terminal domain in addition to the one found in the central region.

Identification and characterization of Iporin as a novel interaction partner for rab1

BackgroundThe small GTPase rab1a and its isoform rab1b are essential regulating components in the vesicle transport between the ER and the Golgi apparatus. Rab1 is thought to act as a molecular switch and can change between an active GTP-bound and an inactive GDP-bound conformation. To elucidate the function of rab1, several approaches have been established to isolate effector proteins, which interact with the activated conformation of rab1. To date p115, GM130, golgin-84 and MICAL have been identified as direct interacting partners. Together with rab1, these molecules are components of a protein complex, which mediates and regulates intracellular vesicle transport.ResultsHere, we report the characterization of Iporin, which is similar to KIAA0375 as a novel rab1-interacting protein. It was initially identified by yeast two-hybrid screening experiments with the active mutant of rab1b (rab1b Q67R) as bait. Iporin contains a SH3 domain and two polyproline stretches, which are known to play a role in protein/protein interactions. In addition, Iporin encloses a RUN domain, which seems to be a major part of the rab1binding domain (R1BD). Iporin is ubiquitously expressed and immunofluorescence staining displays a cytosolic punctual distribution. Interestingly, we also show that Iporin interacts with another rab1 interacting partner, the GM130 protein.ConclusionOur results demonstrate that Iporin is a potential new interacting partner of rab1. Iporin is different from already identified rab1 interacting proteins concerning protein structure and cellular localization. We conclude that Iporin might function as a link between the targeting of ER derived vesicles, triggered by the rab1 GTPase and a signaling pathway regulated by molecules containing SH3 and/or poly-proline regions. The characterization of this novel intermolecular relation could help to elucidate how vesicles find their way from ER to the Golgi apparatus.

A novel MET-interacting protein shares high sequence similarity with RanBPM, but fails to stimulate MET-induced Ras/Erk signaling

MET is a receptor protein tyrosine kinase for hepatocyte growth factor, a multifunctional cytokine controlling cell growth, morphogenesis, and motility. In our previous study, RanBPM/RanBP9, whose name originated from its ability to interact with Ran, was identified as a MET-interacting protein. RanBPM/RanBP9 activates the Ras/Erk signaling pathway by serving as an adaptor protein of MET to recruit Sos. In this study, we identify a protein sharing a high amino acid sequence identity with RanBPM/RanBP9, especially in its SPRY domain, the region responsible for MET binding. This protein lacks the N-terminal poly-proline and poly-glutamine (Poly-PQ) stretch present in RanBPM/RanBP9 and has less homology with RanBPM/RanBP9 in its mid-region. We subsequently named this protein RanBP10 after demonstrating its interaction with Ran. We show that, like RanBPM/RanBP9, RanBP10 interacts with the tyrosine kinase domain of MET via its SPRY domain and these two proteins can compete with each other to bind to MET. Interestingly, unlike RanBPM/RanBP9, overexpression of RanBP10 cannot induce Erk1/2 phosphorylation and serum response element-luciferase (SRE-LUC) reporter gene expression. More importantly, co-transfection of RanBPM/RanBP9 and RanBP10 significantly represses SRE-LUC reporter gene expression induced by overexpression of RanBPM/RanBP9. Additional binding assays demonstrate that RanBP10 fails to interact with Sos, which explains its inability to activate the Ras/Erk pathway. Furthermore, we show that the N-terminus of RanBPM/RanBP9 with the Poly-PQ stretch is required for recruiting Sos and a truncated RanBPM/RanBP9 lacking this region fails to recruit Sos, indicating that the functional difference between RanBP10 and RanBPM/RanBP9 lies in their sequence difference in their N-termini.

Structure/Function Relationships in the Minicollagen ofHydra Nematocysts

The minicollagens found in the inner layer of the Hydra nematocyst walls are the smallest collagens known with 12-16 Gly-X-Y repeats. Minicollagen-1, the best characterized member of this protein family so far, consists of a central collagen triple helix of 12 nm in length flanked at both ends by a polyproline stretch and a conserved cysteine-rich domain. The cysteine-rich tails are proposed to function in the assembly of soluble minicollagen trimers to high molecular structures by a switch of the disulfide linkage from intramolecular to intermolecular bonds. In this study, we investigate the trimeric nature of minicollagen-1 and its capacity to form disulfide-linked polymers in vitro. A fusion protein of minicollagen-1 with maltose-binding protein is secreted as a soluble trimer with only intrachain and no interchain disulfide bridges as confirmed by melting the collagen triple helix under reducing and non-reducing conditions. The conversion of minicollagen-1 trimers to monomers takes place between 40 and 55 degrees C with the melting point being approximately 45 degrees C. Oxidative reshuffling of the minicollagen-1 trimers leads to the formation of high molecular aggregates, which upon reduction show distinct polytrimeric states. Minicollagen trimers in isolated nematocyst capsules proved to be sensitive to SDS and were engaged in polymeric structures with additional cross-links that were resistant to reducing agent.

T(10;11)‐Acute leukemias with MLL‐AF10 and MLL‐ABI1 chimeric transcripts: Specific expression patterns of ABI1 gene in leukemia and solid tumor cell lines

The recurrent translocation t(10;11) is associated with acute myeloid leukemia (AML). The AF10 gene on chromosome 10 at band p12 and MLL at 11q23 fuse in the t(10;11)(p12;q23). Recently, we have identified ABI1 as a new partner gene for MLL in an AML patient with a t(10;11)(p11.2;q23). The ABI1 is a human homologue of the mouse Abl-interactor 1 (Abi1), encoding an Abl-binding protein. The ABI1 protein exhibits sequence similarity to homeotic genes, and contains several polyproline stretches and a src homology 3 (SH3) domain. To clarify the clinical features of t(10;11)-leukemias, we investigated 6 samples from acute leukemia patients with t(10;11) and MLL rearrangement and detected MLL-AF10 chimeric transcripts in 5 samples and MLL-ABI1 in one. The patient with MLL-ABI1 chimeric transcript is the second case described, thus confirming that the fusion of the MLL and ABI1 genes is a recurring abnormality. Both of the patients with MLL-ABI1 chimeric transcript are surviving, suggesting that these patients have a better prognosis than the patients with MLL-AF10. To investigate the roles of AF10 and ABI1 further, we examined the expression of these genes in various cell lines and fresh tumor samples using the reverse transcriptase-polymerase chain reaction method. Although AF10 was expressed in almost all cell lines similarly, the expression patterns of ABI1 were different between leukemia and solid tumor cell lines, suggesting the distinctive role of each isoform of ABI1 in these cell lines. We also determined the complete mouse Abi1 sequence and found that the sequence matched with human ABI1 better than the originally reported Abi1 sequence. Further functional analysis of the MLL-AF10 and MLL-ABI1 fusion proteins will provide new insights into the leukemogenesis of t(10;11)-AML.

Localization of a mammalian homolog of diaphanous, mDia1, to the mitotic spindle in HeLa cells.

mDia1 is a mammalian homolog of Drosophila diaphanous and works as an effector of the small GTPase Rho. It is a member of the formin homology (FH) proteins and contains the Rho-binding domain and an FH3 region in its N terminus, an FH1 region containing polyproline stretches in the middle and an FH2 region in the C terminus. Several lines of evidence indicate that mDia1 and diaphanous are essential in cytokinesis. mDia1 is present in a large amount in the cytoplasm of both interphase and mitotic cells. Using the instantaneous fixation method that preferentially extracts soluble components, we have analyzed localization of mDia1 in mitotic HeLa cells. Immunocytochemistry using polyclonal anti-mDia1 antibody revealed specific immunofluorescence localized to the mitotic spindle. This localization was seen from prophase to telophase. Western blot analysis also detected anti-mDia1 immunoreactivity in the mitotic spindle fraction isolated from mitotic HeLa cells. Consistently, expression of full-length mDia1 as a fusion protein with green fluorescence protein (GFP) revealed the GFP fluorescence again in the mitotic spindle in HeLa cells. Expression of GFP fusions of various truncated mutants of mDia1 identified that this localization is determined by a 173 amino acid-long sequence between the Rho-binding domain and the FH1 region, which contains the C-terminal part of the FH3 region. Point mutation analysis revealed that Leu(434) and Leu(455) in the FH3 region are essential in localization to the mitotic spindle. Neither electroporation of botulinum C3 exoenzyme nor microinjection of Val14RhoA into mitotic cells affected the localization of endogenous mDia1 to the mitotic spindle, suggesting that mDia1 localizes to the mitotic spindle independent of Rho activity. The present study has thus established the mDia1 localization in the mitotic spindle. This localization suggests a role of mDia1 in the spindle-cleavage furrow interaction during cell division.

In vivo importance of actin nucleotide exchange catalyzed by profilin.

The actin monomer-binding protein, profilin, influences the dynamics of actin filaments in vitro by suppressing nucleation, enhancing nucleotide exchange on actin, and promoting barbed-end assembly. Profilin may also link signaling pathways to actin cytoskeleton organization by binding to the phosphoinositide PIP(2) and to polyproline stretches on several proteins. Although activities of profilin have been studied extensively in vitro, the significance of each of these activities in vivo needs to be tested. To study profilin function, we extensively mutagenized the Saccharomyces cerevisiae profilin gene (PFY1) and examined the consequences of specific point mutations on growth and actin organization. The actin-binding region of profilin was shown to be critical in vivo. act1-157, an actin mutant with an increased intrinsic rate of nucleotide exchange, suppressed defects in actin organization, cell growth, and fluid-phase endocytosis of pfy1-4, a profilin mutant defective in actin binding. In reactions containing actin, profilin, and cofilin, profilin was required for fast rates of actin filament turnover. However, Act1-157p circumvented the requirement for profilin. Based on the results of these studies, we conclude that in living cells profilin promotes rapid actin dynamics by regenerating ATP actin from ADP actin-cofilin generated during filament disassembly.

CDNA Cloning and Mapping of a Novel Islet-Brain/JNK-Interacting Protein

IB1/JIP-1 is a scaffold protein that regulates the c-Jun NH2-terminal kinase (JNK) signaling pathway, which is activated by environmental stresses and/or by treatment with proinflammatory cytokines including IL-1β and TNF-α. The JNKs play an essential role in many biological processes, including the maturation and differentiation of immune cells and the apoptosis of cell targets of the immune system. IB1 is expressed predominantly in brain and pancreatic β-cells where it protects cells from proapoptotic programs. Recently, a mutation in the amino-terminus of IB1 was associated with diabetes. A novel isoform, IB2, was cloned and characterized. Overall, both IB1 and IB2 proteins share a very similar organization, with a JNK-binding domain, a Src homology 3 domain, a phosphotyrosine-interacting domain, and polyacidic and polyproline stretches located at similar positions. The IB2 gene (HGMW-approved symbol MAPK8IP2) maps to human chromosome 22q13 and contains 10 coding exons. Northern and RT-PCR analyses indicate that IB2 is expressed in brain and in pancreatic cells, including insulin-secreting cells. IB2 interacts with both JNK and the JNK-kinase MKK7. In addition, ectopic expression of the JNK-binding domain of IB2 decreases IL-1β-induced pancreatic β-cell death. These data establish IB2 as a novel scaffold protein that regulates the JNK signaling pathway in brain and pancreatic β-cells and indicate that IB2 represents a novel candidate gene for diabetes.

Sequential assignment of proline-rich regions in proteins: application to modular binding domain complexes.

Many protein-protein interactions involve amino acid sequences containing proline-rich motifs and even polyproline stretches. The lack of amide protons in such regions complicates assignment, since 1HN-based triple-resonance assignment strategies cannot be employed. Two such systems that we are currently studying include an SH2 domain from the protein Crk with a region containing 9 prolines in a 14 amino acid sequence, as well as a WW domain that interacts with a proline-rich target. A modified version of the HACAN pulse scheme, originally described by Bax and co-workers [Wang et al. (1995) J. Biomol. NMR, 5, 376-382], and an experiment which correlates the intra-residue 1Halpha, 13Calpha/13Cbeta chemical shifts with the 15N shift of the subsequent residue are presented and applied to the two systems listed above, allowing sequential assignment of the molecules.