LIM-only protein FHL3 interacts with CDC25B2 phosphatase

LIM domain proteins are important regulators of the growth, determination, and differentiation of cells. In this report, FHL3 (human four-and-a-half LIM-only protein 3) is shown to interact with human phosphatase CDC25B, a cell cycle regulator involved in the control of G2/M. We found that this interaction was specific to the CDC25B2 isoform. Deletion and point mutation studies indicated that the second LIM domain of FHL3 was essential for this interaction. FRET experiments in C2C12 cells showed that, although both proteins were colocated in the cytoplasm and the nucleus, they interacted only in the nucleus. Finally, we showed that FHL3 binding impaired neither CDC25B2 phosphatase activity nor its localization. Further work is now needed to elucidate the consequences of this interaction on myoblast fate decision and cycle control.     

Insulin-like growth factor binding protein-5 interacts with the vitamin D receptor and modulates the vitamin D response in osteoblasts

The 1,25 dihydroxyvitamin D3 [1,25(OH)2D3]-induced differentiation of osteoblasts comprises the sequential induction of cell cycle arrest at G0/G1 and the expression of bone matrix proteins. Reports differ on the effects of IGF binding protein (IGFBP)-5 on bone cell growth and osteoblastic function. IGFBP-5 can be growth stimulatory or inhibitory and can enhance or impair osteoblast function. In previous studies, we have shown that IGFBP-5 localizes to the nucleus and interacts with the retinoid receptors. We now show that IGFBP-5 interacts with nuclear vitamin D receptor (VDR) and blocks retinoid X receptor (RXR):VDR heterodimerization. VDR and IGFBP-5 were shown to colocalize to the nuclei of MG-63 and U2-OS cells and coimmunoprecipitate in nuclear extracts from these cells. Induction of osteocalcin promoter activity and alkaline phosphatase activity by 1,25(OH)2D3 were significantly enhanced when IGFBP-5 was down-regulated in U2-OS cells. Moreover, we found IGFBP-5 increased basal alkaline phosphatase activity and collagen alpha1 type 1 expression, and that 1,25(OH)2D3 was unable to further induce the expression of these bone differentiation markers in MG-63 cells. Expression of IGFBP-5 inhibited MG-63 cell growth and caused cell cycle arrest at G0/G1 and G2/M. Furthermore, IGFBP-5 reduced the effects of 1,25(OH)2D3 in blocking cell cycle progression at G0/G1 and decreased the expression of cyclin D1. These results demonstrate that IGFBP-5 can interact with VDR to prevent RXR:VDR heterodimerization and suggest that IGFBP-5 may attenuate the 1,25(OH)2D3-induced expression of bone differentiation markers while having a modest effect on the 1,25(OH)2D3-mediated inhibition of cell cycle progression in bone cells.     

Interaction of the heart-specific LIM domain protein, FHL2, with DNA-binding nuclear protein, hNP220

Using a yeast two-hybrid library screen, we have identified that the heart specific FHL2 protein, four-and-a-half LIM protein 2, interacted with human DNA-binding nuclear protein, hNP220. Domain studies by the yeast two-hybrid interaction assay revealed that the second LIM domain together with the third and the fourth LIM domains of FHL2 were responsible to the binding with hNP220. Using green fluorescent protein (GFP)-FHL2 and blue fluorescent protein (BFP)-hNP220 fusion proteins co-expressed in the same cell, we demonstrated a direct interaction between FHL2 and hNP220 in individual nucleus by two-fusion Fluorescence Resonance Energy Transfer (FRET) assay. Besides, Western blot analysis using affinity-purified anti-FHL2 antipeptide antibodies confirmed a 32-kDa protein of FHL2 in heart only. Virtually no expression of FHL2 protein was detected in brain, liver, lung, kidney, testis, skeletal muscle, and spleen. Moreover, the expression of FHL2 protein was also detectable in the human diseased heart tissues. Our results imply that FHL2 protein can shuttle between cytoplasm and nucleus and may act as a molecular adapter to form a multicomplex with hNP220 in the nucleus, thus we speculate that FHL2 may be particularly important for heart muscle differentiation and the maintenance of the heart phenotype.     

PHF5A represents a bridge protein between splicing proteins and ATP-dependent helicases and is differentially expressed during mouse spermatogenesis

PHF5A is a highly conserved protein from yeast to man, and based on studies in yeast, it was suggested that the homologous protein RDS3P in S. cerevisiae takes part in the organization of U2 snRNP particles. By using the yeast two-hybrid assay we could demonstrate that PHF5A interacted both with ATP-dependent helicases EP400 and DDX1 and with arginine-serine (RS)-rich domains of splicing factors U2AF1 and SFRS5 in mouse. Furthermore, domain interaction studies revealed that PHF5A interaction with EP400 and DDX1 is restricted to the N-terminal part of PHF5A, whereas the C-terminal region of PHF5A was found to be responsible for the association with U2AF1 and SFRS5. By using the yeast three-hybrid assay, we could further show that both EP400 and DDX1 interacted only indirectly with U2AF1 and SFRS5 proteins via the bridge protein PHF5A. The subcellular localization of a PHF5A-GFP fusion protein was predominantly observed in the nucleus and, in addition, PHF5A co-localized with both U2AF1 and SFRS5 proteins in nuclear speckles of NIH3T3 cells. Moreover, expression analyses demonstrated that PHF5A and U2AF1 gene expression coincided in spermatocytes during murine spermatogenesis and interaction between these proteins was also detectable in the spermatocyte-specific cell line GC-4spc by using in vivo co-immunoprecipitation studies. Taken together, our results indicate that PHF5A resembles a protein which interacts with splicing factors U2AF1 and SFRS5 and helicases EP400 and DDX1 and functions as a bridge protein between these proteins.     

Potential involvement of the interaction between insulin-like growth factor binding protein (IGFBP)-6 and LIM mineralization protein (LMP)-1 in regulating osteoblast differentiation

Insulin-like growth factor binding protein (IGFBP)-6 has been reported to inhibit differentiation of myoblasts and osteoblasts. In the current study, we explored the mechanisms underlying IGFBP-6 effects on osteoblast differentiation. During MC3T3-E1 osteoblast differentiation, we found that IGFBP-6 protein was down-regulated. Overexpression of IGFBP-6 in MC3T3-E1 and human bone cells inhibited nodule formation, osteocalcin mRNA expression and ALP activity. Furthermore, accumulation of IGFBP-6 in the culture media was not required for any of these effects suggesting that IGFBP-6 suppressed osteoblast differentiation by an intracellular mechanism. A yeast two-hybrid screen of an osteosarcoma library was conducted to identify intracellular binding partners to account for IGFBP-6 inhibitory effects on osteoblast differentiation. LIM mineralizing protein (LMP-1) was identified as a high affinity IGFBP-6 binding partner. Physical interaction between IGFBP-6 and LMP-1 was confirmed by co-immunoprecipitation. Fluorescent protein fusion constructs for LMP-1 and IGFBP-6 were transiently transfected into osteoblasts to provide evidence of subcellular locations for each protein. Coexpression of LMP-1-GFP and IGFBP-6-RFP resulted in overlapping subcellular localization of LMP-1 and IGFBP-6. To determine if there was a functional association of IGFBP-6 and LMP-1 as well as a physical association, we studied the effect of IGFBP-6, LMP-1 and their combination on type I procollagen promoter activity. LMP-1 increased promoter activity while IGFBP-6 reduced promoter activity, and coexpression of LMP-1 with IGFBP-6 abrogated IGFBP-6 suppression. These studies provide evidence that overexpression of IGFBP-6 suppresses human and murine osteoblast differentiation, that IGFBP-6 and LMP-1 physically interact, and supports the conclusion that this interaction may be functionally relevant.     

The four and a half LIM domain protein 2 interacts with and regulates the HERG channel

Protein-protein interactions are critical for protein trafficking, localization and the regulation of ion channels. The human ether-a-go-go-related gene (herg) encodes the alpha-subunit of the potassium channel underlying the rapid component of the cardiac delayed rectifier current. To identify the cellular proteins involved in the regulation of the HERG channel, a human heart cDNA library was screened using a yeast two-hybrid system, with the N-terminus of HERG as bait. The four and a half LIM domain protein 2 (FHL2) was identified as a potential HERG partner. The interaction between these two proteins was confirmed by co-immunoprecipitation and glutathione transferase pull-down assays and immunocytochemical analysis. The physiological implication of HERG-FHL2 interaction, assessed by whole-cell, patch-clamp electrophysiology experiments, showed a significant increase in the HERG current amplitude and a faster deactivation of the tail current in human embryonic kidney 293 cells co-expressing HERG and FHL2. These data indicate that FHL2 interacts with and regulates the HERG channel. Our findings may aid in the further understanding of the molecular basis of HERG channel diversity and arrhythmogenesis in the long-QT syndrome.     

Insulin-like growth factor-binding protein-5 inhibits osteoblast differentiation and skeletal growth by blocking insulin-like growth factor actions

Signaling through the IGF-I receptor by locally synthesized IGF-I or IGF-II is critical for normal skeletal development and for bone remodeling and repair throughout the lifespan. In most tissues, IGF actions are modulated by IGF-binding proteins (IGFBPs). IGFBP-5 is the most abundant IGFBP in bone, and previous studies have suggested that it may either enhance or inhibit osteoblast differentiation in culture and may facilitate or block bone growth in vivo. To resolve these contradictory observations and discern the mechanisms of action of IGFBP-5 in bone, we studied its effects in differentiating osteoblasts and in primary bone cultures. Purified wild-type (WT) mouse IGFBP-5 or a recombinant adenovirus expressing IGFBP-5WT each prevented osteogenic differentiation induced by the cytokine bone morphogenetic protein (BMP)-2 at its earliest stages without interfering with BMP-mediated signaling, whereas an analog with reduced IGF binding (N domain mutant) was ineffective. When added at later phases of bone cell maturation, IGFBP-5WT but not IGFBP-5N blocked mineralization, prevented longitudinal growth of mouse metatarsal bones in short-term primary culture, and inhibited their endochondral ossification. Because an IGF-I variant (R3IGF-I) with diminished affinity for IGFBPs promoted full osteogenic differentiation in the presence of IGFBP-5WT, our results show that IGFBP-5 interferes with IGF action in osteoblasts and provides a framework for discerning mechanisms of collaboration between signal transduction pathways activated by BMPs and IGFs in bone.     

Protein-protein interaction of FHL3 with FHL2 and visualization of their interaction by green fluorescent proteins (GFP) two-fusion fluorescence resonance energy transfer (FRET)

LIM domain proteins are found to be important regulators in cell growth, cell fate determination, cell differentiation and remodeling of the cell cytoskeleton. Human Four-and-a-half LIM-only protein 3 (FHL3) is a type of LIM-only protein that contains four tandemly repeated LIM motifs with an N-terminal single zinc finger (half LIM motif). FHL3 expresses predominantly in human skeletal muscle. In this report, FHL3 was shown to be a novel interacting partner of FHL2 using the yeast two-hybrid assay. Furthermore, site-directed mutagenesis of FHL3 indicated that the LIM2 of FHL3 is the essential LIM domain for interaction with FHL2. Green fluorescent protein (GFP) was used to tag FHL3 in order to study its distribution during myogenesis. Our result shows that FHL3 was localized in the focal adhesions and nucleus of the cells. FHL3 mainly stayed in the focal adhesion during myogenesis. Moreover, using site-directed mutagenesis, the LIM1 of FHL3 was identified as an essential LIM domain for its subcellular localization. Mutants of GFP have given rise to a novel technique, two-fusion fluorescence resonance energy transfer (FRET), in the determination of protein-protein interaction at particular subcellular locations of eukaryotic cells. To determine whether FHL2 and FHL3 can interact with one another and to locate the site of this interaction in a single intact mammalian cell, we fused FHL2 and FHL3 to different mutants of GFP and studied their interactions using FRET. BFP/GFP fusion constructs were cotransfected into muscle myoblast C2C12 to verify the colocalization and subcellular localization of FRET. We found that FHL2 and FHL3 were colocalized in the mitochondria of the C2C12 cells and FRET was observed by using an epi-fluorescent microscope equipped with an FRET specific filter set.     

Insulin-like growth factor binding proteins localize to discrete cell culture compartments in periosteal and osteoblast cultures from fetal rat bone

Insulin-like growth factor (IGF)-I and IGF-II are expressed at biologically effective levels by bone cells. Their stability and activity are modulated by coexpression of IGF binding proteins (IGFBPs). Secreted IGFBPs may partition to soluble, cell-associated, and matrix-bound compartments. Extracellular localization may sequester, store, or present IGFs to appropriate receptors. Of the six IGFBPs known, rat osteoblasts synthesize all but IGFBP-1. Of these, IGFBP-3, -4, and -5 mRNAs are induced by an increase in cAMP. Little is known about extracellular IGFBP localization in bone and nothing about IGFBP expression by nonosteoblastic periosteal bone cells. We compared basal IGFBP expression in periosteal and osteoblast bone cell cultures and assessed the effects of changes in cAMP-dependent protein kinase A or protein kinase C. Basal IGFBP gene expression differed principally in that more IGFBP-2 and -5 occurred in osteoblast cultures, and more IGFBP-3 and -6 occurred in periosteal cultures. An increase in cAMP enhanced IGFBP-3, -4, and -5 mRNA and accordingly increased soluble IGFBP-3, -4, and -5 and matrix-bound IGFBP-3 and -5 in both bone cell populations. In contrast, protein kinase C activators suppressed IGFBP-5 mRNA, and its basal protein levels remained very low. We also detected low M_r bands reactive with antisera to IGFBP-2, -3, and -5, suggesting proteolytic processing or degradation. Our studies reveal that various bone cell populations secrete and bind IGFBPs in selective ways. Importantly, inhibitory IGFBP-4 does not significantly accumulate in cell-associated compartments, even though its secretion is enhanced by cAMP. Because IGFBPs bind IGFs less tightly in cell-bound compartments, they may prolong anabolic effects by agents that increase bone cell cAMP. J. Cell. Biochem. 71:351–362, 1998. © 1998 Wiley-Liss, Inc.     

Structural basis for the bifunctionality of the U5 snRNP 52K protein (CD2BP2)

The bifunctional protein U5-52K is associated with the spliceosomal 20 S U5 snRNP, and it also plays a role in immune response as CD2 receptor binding protein 2 (CD2BP2). U5-52K binds to the CD2 receptor via its GYF-domain specifically recognizing a proline-rich motif on the cytoplasmic surface of the receptor. The GYF-domain is also mediating the interaction of the proteins U5-52K and U5-15K within the spliceosomal U5 snRNP. Here we report the crystal structure of the complex of GYF-domain and U5-15K protein revealing the structural basis for the bifunctionality of the U5-52K protein. The complex structure unveils novel interaction sites on both proteins, as neither the polyproline-binding site of the GYF-domain nor the common ligand-binding cleft of thioredoxin-like proteins, to which U5-15K belongs, are involved in the interaction of U5-15K and U5-52K.     

Serotype-specific Differences in Dengue Virus Non-structural Protein 5 Nuclear Localization

The four serotypes of dengue virus (DENV-1 to -4) cause the most important arthropod-borne viral disease of humans. DENV non-structural protein 5 (NS5) contains enzymatic activities required for capping and replication of the viral RNA genome that occurs in the host cytoplasm. However, previous studies have shown that DENV-2 NS5 accumulates in the nucleus during infection. In this study, we examined the nuclear localization of NS5 for all four DENV serotypes. We demonstrate for the first time that there are serotypic differences in NS5 nuclear localization. Whereas the DENV-2 and -3 proteins accumulate in the nucleus, DENV-1 and -4 NS5 are predominantly if not exclusively localized to the cytoplasm. Comparative studies on the DENV-2 and -4 NS5 proteins revealed that the difference in DENV-4 NS5 nuclear localization was not due to rapid nuclear export but rather the lack of a functional nuclear localization sequence. Interaction studies using DENV-2 and -4 NS5 and human importin-α isoforms failed to identify an interaction that supported the differential nuclear localization of NS5. siRNA knockdown of the human importin-α isoform KPNA2, corresponding to the murine importin-α isoform previously shown to bind to DENV-2 NS5, did not substantially affect DENV-2 NS5 nuclear localization, whereas knockdown of importin-β did. The serotypic differences in NS5 nuclear localization did not correlate with differences in IL-8 gene expression. The results show that NS5 nuclear localization is not strictly required for virus replication but is more likely to have an auxiliary function in the life cycle of specific DENV serotypes.     

Insulin-like growth factor-binding protein-5 (IGFBP-5) stimulates growth and IGF-I secretion in human intestinal smooth muscle by Ras-dependent activation of p38 MAP kinase and Erk1/2 pathways

Insulin-like growth factor-binding protein-5 (IGFBP-5) and insulin-like growth factor-I (IGF-I) are produced by human intestinal smooth muscle cells. Endogenous IGF-I stimulates growth and increases IGFBP-5 secretion. IGFBP-5 augments the effects of IGF-I by facilitating interaction of IGF-I with the IGF-I receptor tyrosine kinase. Andress (Andress, D. L. (1998) Am. J. Physiol. 274, E744-E750) and Berfield et al. (Berfield, A. K., Andress, D. L., and Abrass, C. K. (2000) Kidney Int. 57, 1991-2003) have shown that in osteoblasts and kidney mesangial cells, IGFBP-5 stimulates proliferation and filopodia formation independently of IGF-I, presumably by activating a distinct IGFBP-5 receptor serine kinase. The present study determined whether IGFBP-5 exerts direct effects on growth in human intestinal smooth muscle cells and identified the intracellular signaling pathways involved. IGFBP-5 caused a concentration-dependent increase in [(3)H]thymidine incorporation and an increase in IGF-I secretion that occurred independently of IGF-I and the IGF-I receptor tyrosine kinase. IGFBP-5-induced phosphorylation of p38 MAP kinase, which was abolished by SB203580, or expression of a dominant negative Ras mutant, Ras(S17N), and phosphorylation of Erk1/2, which was abolished by a Raf1 kinase inhibitor, U1026, or expression of Ras(S17N). IGFBP-5-stimulated [(3)H]thymidine incorporation and IGF-I secretion were partly inhibited by SB203580 or U1026 and abolished by the combination of the two inhibitors or by expression of Ras(S17N). These data show that IGFBP-5 stimulates growth and IGF-I secretion in human intestinal smooth muscle cells by activation of p38 MAP kinase-dependent and Erk1/2-dependent pathways that are independent of IGF-I. A positive feedback mechanism therefore links IGFBP-5 and IGF-I secretion that reinforces their individual effects on growth.     

A Cdc2-related protein kinase hPFTAIRE1 from human brain interacting with 14-3-3 proteins

hPFTAIRE1 （PFTK1）, a Cdc2-related protein kinase, is highly expressed in human brain. It exhibits cytoplasmic distribution in Hela cells, although it contains two nuclear localization signals （NLSs） in its N-terminus. To search for its substrates and regulatory components, we screened a two-hybrid library by using the full-length hPFTAIRE1 as a bait. Four 14-3-3 isoforms （β,ε,η,τ） were identified interacting with the hPFTAIRE1. We found a putative 14-3-3 binding consensus motif（RHSSPSS） in the hPFTAIRE 1, which overlapped with its second NLS. Deletion of the RHSSPSS motif or substitution of Ser^119 gwithAla in the conserved binding motif abolished the specific interaction between the hPFTAIRE 1 and the 14-3 -3 proteins. The mutant S 120A hPFTAIRE1 also showed a weak interaction to the 14-3-3 proteins. The results suggested that the Ser^119 is crucial for the interaction between hPFTAIREI and the 14-3-3 proteins. All the hPFTAIRE1 mutants distributed in cytoplasm of Hela cells and human neuroblastoma cells （SH-SY5Y） when fused to the C-terminus of a green fluorescent protein （GFP）, indicating that binding with the 14-3-3 proteins does not contribute to the subcellular localization of the hPFTAIRE1, although the binding may be involved in its signaling regulation.