SMAD蛋白家族:一类新的细胞内信号传导蛋白

SMADs是新近发观的一族细胞内信号传导蛋白,包括8个成员,即SMAD1～8。SMAD1、2、3、5和8是一类,它们被TGF-β受体或BMP受体激活而磷酸化,称为受体调节SMAD,传导TGF-β或BMP的信号。SMAD6和7是另一类,它们抑制受体调节SMAD传导信号。SMAD4是第2类,它是受体调节SMAD传导信号的伴侣。受体调节SMAD传导信号必须先与SMAD4结合形成异源复合物,才能进到核中,调节转录活动。本文简要介绍了各成员的特性及作用。     

Possible role of DMP1 in dentin mineralization

Dentin Matrix Protein 1 (DMP1), the essential noncollagenous proteins in dentin and bone, is believed to play an important role in the mineralization of these tissues, although the mechanisms of its action are not fully understood. To gain insight into DMP1 functions in dentin mineralization we have performed immunomapping of DMP1 in fully mineralized rat incisors and in vitro calcium phosphate mineralization experiments in the presence of DMP1. DMP1 immunofluorescene was localized in peritubular dentin (PTD) and along the dentin-enamel boundary. In vitro phosphorylated DMP1 induced the formation of parallel arrays of crystallites with their c-axes co-aligned. Such crystalline arrangement is a hallmark of mineralized collagen fibrils of bone and dentin. Interestingly, in DMP1-rich PTD, which lacks collagen fibrils, the crystals are organized in a similar manner. Based on our findings we hypothesize, that in vivo DMP1 controls the mineral organization outside of the collagen fibrils and plays a major role in the mineralization of PTD.     

The evolutionarily conserved trimeric structure of CutA1 proteins suggests a role in signal transduction

CutA1 are a protein family present in bacteria, plants, and animals, including humans. Escherichia coli CutA1 is involved in copper tolerance, whereas mammalian proteins are implicated in the anchoring of acetylcholinesterase in neuronal cell membranes. The x-ray structures of CutA1 from E. coli and rat were determined. Both proteins are trimeric in the crystals and in solution through an inter-subunit beta-sheet formation. Each subunit consists of a ferredoxin-like (beta1alpha1beta2beta3alpha2beta4) fold with an additional strand (beta5), a C-terminal helix (alpha3), and an unusual extended beta-hairpin involving strands beta2 and beta3. The bacterial CutA1 is able to bind copper(II) in vitro through His2Cys coordination in a type II water-accessible site, whereas the rat protein precipitates in the presence of copper(II). The evolutionarily conserved trimeric assembly of CutA1 is reminiscent of the architecture of PII signal transduction proteins. This similarity suggests an intriguing role of CutA1 proteins in signal transduction through allosteric communications between subunits.     

Nuclear localization of herpesvirus proteins: potential role for the cellular framework.

Two herpes simplex virus proteins, the major capsid protein and the major DNA binding protein, are specifically localized to the nucleus of infected cells. We have found that the major proportion of these proteins is associated with the detergent-insoluble matrix or cytoskeletal framework of the infected cell from the time of their synthesis until they have matured to their final binding site in the cell nucleus. These results suggest that these two proteins may interact with or bind to the cellular cytoskeleton during or soon after their synthesis and throughout transport into the cell nucleus. In addition, the DNA binding protein remains associated with the nuclear skeleton at times when it is bound to viral DNA. Thus, viral DNA may also be attached to the nuclear framework. We have demonstrated that the DNA binding protein and the capsid protein exchange from the cytoplasmic framework to the nuclear framework, suggesting the direct movement of the proteins from one structure to the other. Inhibition of viral DNA replication enhanced the binding of the DNA binding protein to the cytoskeleton and increased the rate of exchange from the cytoplasmic framework to the nuclear framework, suggesting a functional relationship between these events. Inhibition of viral DNA replication resulted in decreased synthesis and transport of the capsid protein. We have been unable to detect any artificial binding of these proteins to the cytoskeleton when solubilized viral proteins were mixed with a cytoskeletal fraction or a cell monolayer. This suggested that the attachment of these proteins to the cytoskeleton represents the actual state of these proteins within the cell.     

TAB-1 modulates intracellular localization of p38 MAP kinase and downstream signaling

Stress-activated mitogen-activated protein (MAP) kinase p38 mediates stress signaling in mammalian cells via threonine and tyrosine phosphorylation in its conserved TGY motif by upstream MAP kinase kinases (MKKs). In addition, p38 MAP kinase can also be activated by an MKK-independent mechanism involving TAB-1 (TAK-1-binding protein)-mediated autophosphorylation. Although TAB-1-mediated p38 activation has been implicated in ischemic heart, the biological consequences and downstream signaling of TAB-1-mediated p38 activation in cardiomyocytes is largely unknown. We show here that TAB-1 expression leads to a significant induction of p38 autophosphorylation and consequent kinase activation in cultured neonatal cardiomyocytes. In contrast to MKK3-induced p38 kinase downstream effects, TAB-1-induced p38 kinase activation does not induce expression of pro-inflammatory genes, cardiac marker gene expression, or changes in cellular morphology. Rather, TAB-1 binds to p38 and prevents p38 nuclear localization. Furthermore, TAB-1 disrupts p38 interaction with MKK3 and redirects p38 localization in the cytosol. Consequently, TAB-1 expression antagonizes the downstream activity of p38 kinase induced by MKK3 and attenuates interleukin-1beta-induced inflammatory gene induction in cardiomyocytes. These data suggest that TAB-1 can mediate MKK-independent p38 kinase activation while negatively modulating MKK-dependent p38 function. Our study not only redefines the functional role of TAB-1 in p38 kinase-mediated signaling pathways but also provides the first evidence that intracellular localization of p38 kinase and complex interaction dictates its downstream effects. These results suggest a previously unknown mechanism for stress-MAP kinase regulation in mammalian cells.     

Nuclear localization and the heat shock proteins

The highly conserved heat shock proteins (HSP) belong to a subset of cellular proteins that localize to the nucleus. HSPs are atypical nuclear proteins in that they localize to the nucleus selectively, rather than invariably. Nuclear localization of HSPs is associated with cell stress and cell growth. This aspect of HSPs is highly conserved with nuclear localization occurring in response to a wide variety of cell stresses. Nuclear localization is likely important for at least some of the heat shock proteins’ protective functions; little is known about the function of the heat shock proteins in the nucleus. Nuclear localization is signalled by the presence of a basic nuclear localization sequence (NLS) within a protein. Though most is known about HSP 72’s nuclear localization, the NLS(s) has not been definitively identified for any of the heat shock proteins. Likely more is involved than presence of a NLS; since the heat shock proteins only localize to the nucleus under selective conditions, nuclear localization must be regulated. HSPs also function as chaperons of nuclear transport, facilitating the movement of other macromolecules across the nuclear membrane. The mechanisms involved in chaperoning of proteins by HSPs into the nucleus are still being identified.     

The role of mechanical stretching in the activation and localization of adhesion proteins and related intracellular molecules

The molecular complexity of the processes which lead to cell adhesion includes membrane and cytoskeletal proteins, involved in the focal adhesion formation, as well as signaling molecules tightly associated with the main intracellular regulatory cascades (Akt/PKB and MAPK/Erk). Dynamic environments, which create substrate deformations at determined frequencies and timing, have significant influences on adhesion mechanisms and in general in cellular behavior. In this work, we investigated the role of mechanical stretching (10% substrate deformation, 1 Hz frequency applied up to 60 min) on adhesion proteins (vinculin and focal adhesion kinase—FAK), related RhoGTPases (Rac1 and RhoA), and intracellular pathways (Akt/PKB and MAPK/Erk) in terms of activation and membrane recruitment in relation with cytoskeletal changes observed (membrane ruffling and filopodia formation). These changes are due to intracellular molecular rearrangements, acting with sequential concerted dynamics, able to modify the cytoskeletal conformation. The observed cellular response adds some important issues for better understanding the cellular behavior in environment which mimic as close as possible the physiological conditions. J. Cell. Biochem. 112: 1403–1409, 2011. © 2011 Wiley‐Liss, Inc.     

Immunodetection and intracellular localization of caldesmon-like proteins in Amoeba proteus

Summary. Caldesmon immunoanalogues were detected in Amoeba proteus cell homogenates by the Western blot technique. Three immunoreactive bands were recognized by polyclonal antibodies against the whole molecule of chicken gizzard caldesmon as well as by a monoclonal antibody against its C-terminal domain: one major and two minor bands corresponding to proteins with apparent molecular masses of 150, 69, and 60 kDa. The presence of caldesmon-like protein(s) in amoebae was revealed as well in single cells after their fixation, staining with the same antibodies, and recording their total fluorescence in a confocal laser scanning microscope. Proteins recognized by the antibodies bind to filamentous actin. This was established by a cosedimentation assay in cell homogenates and by colocalization of the caldesmon-related immunofluorescence with the fluorescence of filamentous actin stained with rhodamine-labelled phalloidin, demonstrated in optical sections of single cells in a confocal microscope. Caldesmon is colocalized with filamentous actin in the withdrawn cell regions where the cortical actomyosin network contracts and actin is depolymerized, in the frontal zone where actin is polymerized again and the cortical cytoskeleton is reconstructed, inside the nucleus and in the perinuclear cytoskeleton, and probably at the cell-to-substratum adhesion sites. The regulatory role of caldesmon in these functionally different regions of locomoting amoebae is discussed.Correspondence and reprints: Department of Cell Biology, Nencki Institute of Experimental Biology, ulica Pasteura 3, 02-093 Warsaw, Poland.Received October 7, 2002; accepted December 2, 2002; published online August 26, 2003     

Nuclear localization signal and phosphorylation of Serine350 specify intracellular localization of DRAK2

DAP kinase-related apoptosis-inducing kinase 2 (DRAK2) is a serine/threonine kinase of the death-associated protein kinase family. DRAK2 mediates apoptosis induced by extracellular stimuli, including UV irradiation and interleukin-2, and also regulates T-cell receptor sensitivity in developing thymocytes. During these events, the subcellular localization of DRAK2 changes between the nucleus and cytoplasm. We found that DRAK2 has a putative nuclear-localization signal (NLS) sequence. Mutations in this sequence interfered with DRAK2 localization to the nucleus. Furthermore, green fluorescence protein fused to the putative NLS accumulated in the nucleus, indicating that the putative sequence functions as an NLS. We also found that the function of the NLS was regulated by phosphorylation. Phorbol myristate acetate (PMA) induced the accumulation of DRAK2 in the cytoplasm of NIH3T3 cells, whereas in the absence of PMA, DRAK2 was localized to the nucleus. Ectopic expression of PKC-gamma induced cytoplasmic localization of DRAK2 and PKC-gamma phosphorylated Ser350 flanking the NLS. DRAK2, but not the Ser350Asp mutant, accumulated in the nuclei of ACL-15 cells in response to UV-irradiation. These results suggest that phosphorylation of Ser350 plays an essential role in regulating translocation of DRAK2 to the nucleus from the cytoplasm, possibly by affecting the activity of the NLS.     

Study of a temperature-sensitive mutant of the ras-related YPT1 gene product in yeast suggests a role in the regulation of intracellular calcium

Intragenic mutations were isolated that suppressed the dominant-lethal phenotype of the YPT1ile121 mutant gene in a temperature-dependent fashion. Among different amino acid substitutions resulting from single point mutations, two, Ala161----Val (A161V) and Met165----Ile (M165I), restored the function of the YPT1ile121 mutant protein. Mutants expressing the YPT1ile121/val161 allele (ypt1ts) only, grew normally at temperatures up to 30 degrees C but were arrested at 37 degrees C. At the restrictive temperature, ypt1ts mutants accumulated ER membranes, small vesicles, and unprocessed invertase, and they exhibited cytoskeletal defects and an enhanced 45Ca2+ uptake. Similar alterations were seen in YPT1-depleted cells. The ypt1ts mutant cells could be rescued from growth arrest by increasing extracellular Ca2+, and, even at the permissive temperature, they displayed increased trifluoperazine sensitivity.     

Comparison of intracellular localization of Nubp1 and Nubp2 using GFP fusion proteins

Nubp1 (also known as Nbp35) and Nubp2 (also known as Cfd1) proteins are known to be responsible for regulating centrosome duplication in mouse and ribosome biogenesis in yeast. Nubp proteins contribute to diverse physiological functions. It is thought that Nubp1 and Nubp2 proteins interact with each other and regulate their functions. However, little is known about the intracellular localization of Nubp proteins. In this study, we compared the intracellular localization of human Nubp1 and Nubp2 by fusing these proteins with green fluorescent protein (GFP) in HeLa cells. The nuclear transfer of Nubp1–GFP, where GFP was fused to the C-terminus, was not observed. However, GFP–Nubp1, where GFP was fused to the N-terminus, did accumulate in the nucleus. In addition, GFP-modification at the N-terminal of Nubp2 induced nuclear transformation. Our data suggest that the C-terminal region of Nubp1 is important for nuclear transfer and the N-terminal of Nubp2 contributes to the morphology of the nucleus.     

Fatty acylated proteins as components of intracellular signaling pathways

From the studies presented above, it is obvious that fatty acylation is a common modification among proteins involved in cellular regulatory pathways, and in certain cases mutational analyses have demonstrated the importance of covalent fatty acids in the functioning of these proteins. Indeed, certain properties provided by fatty acylation make it an attractive modification for regulatory proteins that might interact with many different substrates, particularly those found at or near the plasma membrane/cytosol interface. In the case of intracellular fatty acylated proteins, the fatty acyl moiety allows tight binding to the plasma membrane without the need for cotranslational insertion through the bilayer. For example, consider the tight, salt-resistant interaction of myristoylated SRC with the membrane, whereas its nonmyristoylated counterpart is completely soluble. Likewise for the RAS proteins, which associate weakly with the membrane in the absence of fatty acylation, while palmitoylation increases their affinity for the plasma membrane and their biological activity. Fatty acylation also permits reversible membrane association in some cases, particularly for several myristoylated proteins, thus conferring plasticity on their interactions with various signaling pathway components. Finally, although this has not been demonstrated, it is conceivable that covalent fatty acid may allow for rapid mobility of proteins within the membrane. Several questions remain to be answered concerning requirements for fatty acylation by regulatory proteins. The identity of the putative SRC "receptor" will provide important clues as to the pathways in which normal SRC functions, as well as into the process of transformation by oncogenic tyrosine kinases. The possibility that other fatty acylated proteins associate with the plasma membrane in an analogous manner also needs to be investigated. An intriguing observation that can be made from the information presented here is that at least three different families of proteins involved in growth factor signaling pathways encode both acylated and nonacylated members, suggesting that selective fatty acylation may provide a means of determining the specificity of their interactions with other regulatory molecules. Further studies of fatty acylated proteins should yield important information concerning the regulation of intracellular signaling pathways utilized during growth and differentiation.     

Nuclear lipid signaling: novel role of eicosanoids

Nuclear lipid signaling is an established, widespread mechanism that operates in multiple cellular processes including proliferative and differentiative responses to a variety of stimuli. In this literature review with key references highlighted, we put forward the hypothesis that differential flow through various intracrine mechanisms can dictate resultant cellular actions such as mitosis, differentiation, or apoptosis.