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1: Biochemical and biophysical research communications, 2008 Apr 18, 368(4)
URP2SF, a FERM and PH domain containing protein, regulates NF-kappaB and apoptosis.

[Abstract]The transcription factor NF-kappaB plays an important role in inflammation and cell survival. Here, we report the functional characterization of URP2SF (UNC-112 related protein 2 short form), which is a novel protein containing FERM and PH domains. The protein is conserved in evolution across different species. Northern blot and RT-PCR analysis revealed that the expression of URP2SF appeared to be primarily confined to immune system. URP2SF protein localized to the whole cell. Over-expression of URP2SF strongly repressed NF-kappaB activity in the absence or presence of NF-kappaB stimulus. Importantly, URP2SF over-expression also induced cell apoptosis. Furthermore, suppression of URP2SF expression by small interference RNA significantly activated the NF-kappaB reporter gene activities, as well as attenuated TNF-alpha induced apoptosis. Therefore, our data show for the first time that URP2SF may act as a transcriptional repressor in NF-kappaB signaling pathway and regulate cell apoptotic pathway.

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2: Cellular signalling, 2007 May, 19(5)
The PH domain containing protein CKIP-1 binds to IFP35 and Nmi and is involved in cytokine signaling.

[Abstract]The pleckstrin homology domain-containing protein CKIP-1 is implicated in regulation of cell differentiation, apoptosis, cytoskeleton as well as recruitment of CK2 and ATM kinases to plasma membrane. Protein-protein interactions of CKIP-1 were required for these functions. Here we identify the IFN-induced protein IFP35 and its homologue Nmi as two novel CKIP-1 interacting partners. The NID domains of IFP35 and Nmi are required for the interactions. Similar to IFP35 and Nmi, CKIP-1 can be up-regulated dramatically by IFN-gamma and IL-2 and form homodimer and homotrimer in vivo. Nmi stabilizes IFP35, whereas CKIP-1 destabilizes IFP35 via inhibiting IFP35-Nmi interaction. The ratio of Nmi to CKIP-1 determines the stability of IFP35 and control cytokine signaling in a novel mechanism. Importantly, similar to Nmi and contrast to IFP35, CKIP-1 inhibits tumor cell growth and Akt-mediated cell survival. Thus, our results provide a novel role of CKIP-1 in cytokine signaling response and the biochemical mechanism, by which two previously identified modulators IFP35 and Nmi are involved via interactions.

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3: Journal of cell science, 2006 Mar 15, 119(Pt 6)
Single-molecule analysis of chemoattractant-stimulated membrane recruitment of a PH-domain-containing protein.

[Abstract]Molecular mechanisms of chemotactic response are highly conserved among many eukaryotic cells including human leukocytes and Dictyostelium discoideum cells. The cells can sense the differences in chemoattractant concentration across the cell body and respond by extending pseudopods from the cell side facing to a higher concentration. Pseudopod formation is regulated by binding of pleckstrin homology (PH)-domain-containing proteins to phosphatidylinositol 3,4,5-trisphosphates [PtdIns(3,4,5)P3] localized at the leading edge of chemotaxing cells. However, molecular mechanisms underlying dynamic features of a pseudopod have not been fully explained by the known properties of PH-domain-containing proteins. To investigate the mechanisms, we visualized single molecules of green fluorescent protein tagged to Crac (Crac-GFP), a PH-domain-containing protein in D. discoideum cells. Whereas populations of Crac molecules exhibited a stable steady-state localization at pseudopods, individual molecules bound transiently to PtdIns(3,4,5)P3 for approximately 120 milliseconds, indicating dynamic properties of the PH-domain-containing protein. Receptor stimulation did not alter the binding stability but regulated the number of bound PH-domain molecules by metabolism of PtdIns(3,4,5)P3. These results demonstrate that the steady-state localization of PH-domain-containing proteins at the leading edge of chemotaxing cells is dynamically maintained by rapid recycling of individual PH-domain-containing proteins. The short interaction between PH domains and PtdIns(3,4,5)P3 contributes to accurate and sensitive chemotactic movements through the dynamic redistributions. These dynamic properties might be a common feature of signaling components involved in chemotaxis.

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4: Molecular and cellular biology, 2004 Oct, 24(20)
PHR1, a PH domain-containing protein expressed in primary sensory neurons.

[Abstract]Previously, we identified PHR1 as an abundantly expressed gene in photoreceptors and showed that it encodes four isoforms, each with N-terminal pleckstrin homology (PH) and C-terminal transmembrane domains. To better understand PHR1 function and expression, we made a Phr1 null mouse by inserting a beta-galactosidase/neor cassette into exon 3. In addition to photoreceptors, we found abundant expression of specific Phr1 splice forms in olfactory receptor neurons and vestibular and cochlear hair cells. We also found Phr1 expression in cells with a possible sensory function, including peripheral retinal ganglion cells, cochlear interdental cells, and neurons of the circumventricular organ. Despite this discrete expression in known and putative sensory neurons, mice lacking PHR1 do not have overt sensory deficits.

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5: Biochimie, 2004 Aug, 86(8)
Identification and characterization of Veph, a novel gene encoding a PH domain-containing protein expressed in the developing central nervous system of vertebrates.

[Abstract]We isolated Veph, a novel gene encoding a pleckstrin homology (PH) domain-containing protein from a mouse. Veph was strongly expressed in the embryonic brain, and its expression level gradually decreased in later stages. In situ hybridization analysis of sectioned embryo brains revealed that Veph was expressed exclusively in the ventricular zone. We then isolated a zebrafish orthologue of Veph (zVeph). As observed in the mouse gene, zVeph was expressed in the ventricular zone of developing brain and spinal cord. Blockage of zVeph expression by injection of zVeph-specific morpholino antisense oligo into zebrafish fertilized eggs resulted in a defect in the midbrain-hindbrain boundary and otic vesicle formation, suggesting the important function of zVeph in central nervous system (CNS) development. On the other hand, homozygous knockout mice of Veph showed no significant defect in the CNS, pointing to possible different functions of Veph between the zebrafish and mouse.

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6: The Biochemical journal, 2001 Sep 1, 358(Pt 2)
Crystal structure of the phosphatidylinositol 3,4-bisphosphate-binding pleckstrin homology (PH) domain of tandem PH-domain-containing protein 1 (TAPP1): molecular basis of lipid specificity.

[Abstract]Phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)] and its immediate breakdown product PtdIns(3,4)P(2) function as second messengers in growth factor- and insulin-induced signalling pathways. One of the ways that these 3-phosphoinositides are known to regulate downstream signalling events is by attracting proteins that possess specific PtdIns-binding pleckstrin homology (PH) domains to the plasma membrane. Many of these proteins, such as protein kinase B, phosphoinositide-dependent kinase 1 and the dual adaptor for phosphotyrosine and 3-phosphoinositides (DAPP1) interact with both PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2) with similar affinity. Recently, a new PH-domain-containing protein, termed tandem PH-domain-containing protein (TAPP) 1, was described which is the first protein reported to bind PtdIns(3,4)P(2) specifically. Here we describe the crystal structure of the PtdIns(3,4)P(2)-binding PH domain of TAPP1 at 1.4 A (1 A=0.1 nm) resolution in complex with an ordered citrate molecule. The structure is similar to the known structure of the PH domain of DAPP1 around the D-3 and D-4 inositol-phosphate-binding sites. However, a glycine residue adjacent to the D-5 inositol-phosphate-binding site in DAPP1 is substituted for a larger alanine residue in TAPP1, which also induces a conformational change in the neighbouring residues. We show that mutation of this glycine to alanine in DAPP1 converts DAPP1 into a TAPP1-like PH domain that only interacts with PtdIns(3,4)P(2), whereas the alanine to glycine mutation in TAPP1 permits the TAPP1 PH domain to interact with PtdIns(3,4,5)P(3).

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7: Experimental lung research, 2000 Jun, 26(4)
KPL1, which encodes a novel PH domain-containing protein, is induced during ciliated cell differentiation of rat tracheal epithelial cells.

[Abstract]Using differential display, we have identified a novel gene, KPL1, induced in rat tracheal epithelial (RTE) cells grown under conditions which stimulate ciliogenesis. The KPL1 protein is predicted to contain a pleckstrin homology (PH) domain, which has been found in numerous signal transduction and cytoskeletal proteins. These domains are thought to function by recruiting proteins to cellular membranes, and they have been shown to bind phosphoinositols and the beta/gamma subunit of G proteins. We have cloned rat and human KPL1; the predicted protein translations are 94% identical. Alternate transcripts exist in rat and human tracheal cells that predict a protein which contains a 35-amino acid insert. KPL1 was upregulated in RTE cultures undergoing mucociliary but not squamous differentiation; and in cultures undergoing mucociliary differentiation, KPL1 expression most closely paralleled that of a marker of ciliated cell differentiation (axonemal dynein heavy chain) and not a marker of mucous cell differentiation (mucin 5AC). As a new member of the family of PH domain-containing proteins, KPL1 may have a unique role in ciliated cell differentiation or function.