The inner nuclear membrane protein p58 associates in vivo with a p58 kinase and the nuclear lamins.

p58, also referred to as the lamin B receptor, is an intrinsic protein of the inner nuclear membrane that binds in vitro to lamin B. Previous studies have demonstrated that p58 is phosphorylated in vivo and removal of its phosphate moieties affects lamin B binding. Using affinity-purified antipeptide antibodies, we have now immunoisolated p58 from bird erythrocyte lysates under isotonic, non-denaturing conditions. Analysis of the immunopurified material shows that five distinct proteins are tightly and specifically associated with p58. Two of these polypeptides can be identified as nuclear lamins A and B. The immunoisolate also contains a kinase activity that phosphorylates p58 in vivo and in vitro, exclusively at serine residues, as indicated by phosphoamino acid analysis and two-dimensional phosphopeptide mapping. Cell fractionation experiments and in vitro phosphorylation assays demonstrate that the p58 kinase resides in the nuclear envelope and is distinct from protein kinase A and cdc2 kinase, for both of which p58 is an in vitro substrate. These data suggest that p58 is interacting in vivo with a p58 kinase and the nuclear lamins.     

Small heat shock protein p26 associates with nuclear lamins and HSP70 in nuclei and nuclear matrix fractions from stressed cells

The small heat shock/alpha-crystallin protein p26 undergoes nuclear translocation in response to stress in encysted embryos of the brine shrimp Artemia franciscana. About 50% of total p26 translocates to nuclei in embryos treated with heat shock or anoxia, and in embryo homogenates incubated at low pH. Nuclear fractionation shows that the majority of nuclear p26 and a nuclear lamin are associated with the nuclear matrix fraction. To further explore the roles of p26 and other HSPs in stabilizing nuclear matrix proteins (NMPs), nuclear matrices from control, and heat-shocked embryos were disassembled in urea and evaluated by one and two-dimensional (2-D) gel electrophoresis and Western immunoblotting after reassembling. Nuclear lamins were present only in reassembled fractions and, in the case of heat shock, p26 and HSP70 were also present. HSP90 was not detected in any nuclear fraction. Confocal microscopy on isolated nuclei and nuclear matrix preparations from control and heat-shocked embryos showed that the majority of p26 and a nuclear lamin share similar nuclear distributions. The combination of microscopy and fractionation results suggests that p26 and HSP70 play a role in the protection of nuclear lamins within the nuclear matrix.     

Phosphatidylinositol kinase activity associates with viral p60src protein.

Immunoprecipitates of p60v-src proteins from chicken embryo fibroblasts infected with Rous sarcoma virus were assayed for phosphatidylinositol (PI) kinase activity in the absence of detergents. The product of the PI kinase reaction, phosphatidylinositol monophosphate (PIP), migrated slightly slower than did the authentic phosphatidylinositol-4-monophosphate marker in thin-layer chromatography and was indistinguishable from phosphatidylinositol-3-monophosphate produced by PI kinase type I. Furthermore, the deacylated product comigrated with glycerophosphoinositol-3-phosphate in high-performance liquid chromatography. Both sucrose gradient fractionation and the heat stability of PI kinase activity from cells infected with temperature-sensitive mutants suggest that the PI kinase activity is not intrinsic to p60v-src but is a property of another molecule complexed with p60v-src. All transforming variants of p60src were associated with PI kinase activity, whereas this enzyme activity was hardly detectable in immunoprecipitates from cells infected with nontransforming viruses encoding p60c-src or an enzymatically inactive variant. However, PI kinase activity was found in p60src immunoprecipitates from cells infected with nonmyristylated, nontransforming mutants as well as temperature-sensitive mutants at the nonpermissive temperature, which indicated that simple association of PI kinase activity with p60src is not sufficient for cell transformation.     

The insulator binding protein CTCF associates with the nuclear matrix

Nuclear DNA is organized into chromatin loop domains. At the base of these loops, matrix-associated regions (MARs) of the DNA interact with nuclear matrix proteins. MARs act as structural boundaries within chromatin, and MAR binding proteins may recruit multiprotein complexes that remodel chromatin. The potential tumor suppressor protein CTCF binds to vertebrate insulators and is required for insulator activity. We demonstrate that CTCF is associated with the nuclear matrix and can be cross-linked to DNA by cisplatin, an agent that preferentially cross-links nuclear matrix proteins to DNA in situ. These results suggest that CTCF anchors chromatin to the nuclear matrix, suggesting that there is a functional connection between insulators and the nuclear matrix. We also show that the chromatin-modifying enzymes HDAC1 and HDAC2, which are intrinsic nuclear matrix components and thought to function as corepressors of CTCF, are incapable of associating with CTCF. Hence, the insulator activity of CTCF apparently involves an HDAC-independent association with the nuclear matrix. We propose that CTCF may demarcate nuclear matrix-dependent points of transition in chromatin, thereby forming topologically independent chromatin loops that may support gene silencing.     

Steel factor stimulates the tyrosine phosphorylation of the proto-oncogene product, p95vav, in human hemopoietic cells.

Steel factor (SF) (also called stem cell factor, mast cell growth factor, or c-kit ligand) is a recently cloned hemopoietic growth factor that is produced by bone marrow stromal cells, fibroblasts, and hepatocytes. In both mouse and man it acts synergistically with several colony stimulating factors, including interleukin-3 (IL-3) and granulocyte macrophage-colony stimulating factor (GM-CSF), to induce the proliferation and differentiation of primitive hemopoietic precursor cells. In order to study its mechanism of action and to explore the molecular basis for its synergistic activity we have examined the proteins that become tyrosine phosphorylated in response to SF, IL-3, and GM-CSF. We report herein that SF, but not IL-3 or GM-CSF, dramatically stimulates the tyrosine phosphorylation of the product of the recently discovered proto-oncogene, vav, in two SF-responsive human cell lines, M07E and TF-1. Although phosphorylation is very rapid, reaching maximal levels within 2 min at 37 degrees C, co-immunoprecipitation studies suggest that c-kit may either not associate directly with p95vav or bind to it with very low affinity. Nonetheless, our data suggest that c-kit may utilize p95vav to mediate downstream signaling in hemopoietic cells.     

Nup2p dynamically associates with the distal regions of the yeast nuclear pore complex.

Nucleocytoplasmic transport is mediated by the interplay between soluble transport factors and nucleoporins resident within the nuclear pore complex (NPC). Understanding this process demands knowledge of components of both the soluble and stationary phases and the interface between them. Here, we provide evidence that Nup2p, previously considered to be a typical yeast nucleoporin that binds import- and export-bound karyopherins, dynamically associates with the NPC in a Ran-facilitated manner. When bound to the NPC, Nup2p associates with regions corresponding to the nuclear basket and cytoplasmic fibrils. On the nucleoplasmic face, where the Ran--GTP levels are predicted to be high, Nup2p binds to Nup60p. Deletion of NUP60 renders Nup2p nucleoplasmic and compromises Nup2p-mediated recycling of Kap60p/Srp1p. Depletion of Ran--GTP by metabolic poisoning, disruption of the Ran cycle, or in vitro by cell lysis, results in a shift of Nup2p from the nucleoplasm to the cytoplasmic face of the NPC. This mobility of Nup2p was also detected using heterokaryons where, unlike nucleoporins, Nup2p was observed to move from one nucleus to the other. Together, our data support a model in which Nup2p movement facilitates the transition between the import and export phases of nucleocytoplasmic transport.     

Schizosaccharomyces pombe Mcm3p, an essential nuclear protein, associates tightly with Nda4p (Mcm5p).

MCM proteins are required for the proper regulation of DNA replication. There are six MCM proteins in all eukaryotes which interact to form a large complex. We report the cloning of fission yeast mcm3 +. mcm3 + is essential and spores carrying a Delta mcm3 disruption arrest with an apparently replicated DNA content. The protein is found constitutively in the nucleus and levels remain constant throughout the cell cycle. Mcm3p binds particularly tightly to Nda4p (Mcm5p), but is loosely associated with the other Schizosaccharomyces pombe MCM proteins. Thus, Mcm3p is a peripheral MCM subunit.     

Phospholipase C-gamma 1 directly associates with the p70 trk oncogene product through its src homology domains.

Tyrosine phosphorylation of proteins was examined in NIH3T3 cells transformed by an oncogenic form of the trk protein. Proteins of 148, 140, 70, and 55 kDa were phosphorylated on tyrosine residues in trk-transformed cells but not control NIH3T3 cells. The 70-kDa protein may represent the trk oncogene protein itself which was shown to be tyrosine-phosphorylated in vivo using trk-specific antiserum. Phospholipase C-gamma 1 (PLC-gamma 1) was also found to be constitutively tyrosine-phosphorylated in trk-transformed cells and the trk protein co-immunoprecipitated with PLC-gamma. The GTPase-activating protein of ras (GAP) and the 62-kDa GAP-associated protein were tyrosine-phosphorylated in trk-transformed cells, and a lesser amount of trk co-immunoprecipitated with GAP relative to with PLC-gamma. The trk oncogene product bound specifically to a bacterially expressed fusion protein containing the src homology domains of PLC-gamma. The data suggest a significant role for PLC-gamma in intracellular signaling by the trk oncogene.     

BAG-1 associates with Hsc70.Tau complex and regulates the proteasomal degradation of Tau protein

Intraneuronal accumulation of phosphorylated Tau protein is a molecular pathology found in many forms of dementia, including Alzheimer disease. Research into possible mechanisms leading to the accumulation of modified Tau protein and the possibility of removing Tau protein from the system have revealed that the chaperone protein system can interact with Tau and mediate its degradation. Hsp70/Hsc70, a member of the chaperone protein family, interacts with Tau protein and mediates proper folding of Tau and can promote degradation of Tau protein under certain circumstances. However, because Hsp70/Hsc70 has many binding partners that can mediate its activity, there is still much to discover about how Hsp70 acts in vivo to regulate Tau protein. BAG-1, an Hsp70/Hsc70 binding partner, has been implicated as a mediator of neuronal function. In this work we show that BAG-1 associates with Tau protein in an Hsc70-dependent manner. Overexpression of BAG-1 induced an increase in Tau levels, which is shown to be due to an inhibition of protein degradation. We further show that BAG-1 can inhibit the degradation of Tau protein by the 20 S proteasome but does not affect the ubiquitination of Tau protein. RNA-mediated interference depletion of BAG-1 leads to a decrease in total Tau protein levels as well as promoting hyperphosphorylation of the remaining protein. Induction of Hsp70 by heat shock enhanced the increase of Tau levels in cells overexpressing BAG-1 but induced a decrease of Tau levels in cells that were depleted of BAG-1. Finally, BAG-1 is highly expressed in neurons bearing Tau tangles in a mouse model of Alzheimer disease. This data suggests a molecular mechanism through which Tau protein levels are regulated in the cell and possible consequences for the pathology and treatment of Alzheimer disease.     

SWAP-70 associates transiently with macropinosomes

Cells accomplish the non-selective uptake of extracellular fluids, antigens and pathogens by the endocytic process of macropinocytosis. The protein SWAP-70 is a widely expressed, pleckstrin-homology (PH) domain-containing protein that marks a transitional subset of actin filaments in motile cells. Here we report that the protein SWAP-70 associates transiently with macropinosomes in dendritic cells and NIH/3T3 fibroblasts. The association of SWAP-70 with macropinosomes is preceded by the accumulation of Rac-GTP and followed by that of Rab5. Three regions of SWAP-70, the N-terminal region, the PH domain and the C-terminal region, contribute in a combinatorial manner to the transient association with newly formed macropinosomes in the cell periphery and occasionally with aged macropinosomes on their passage to the cell center. These data identify SWAP-70 as a transient component of early macropinosomes.     

The Epstein-Barr virus nuclear antigen leader protein associates with hsp72/hsc73.

Epstein-Barr virus nuclear antigen leader protein (EBNA-LP) is important for primary B-lymphocyte growth transformation. We now demonstrate that the W repeat-encoded domain of EBNA-LP significantly associates with proteins of the heat shock protein 70 family (hsp72/hsc73). hsp72/hsc73 may mediate the previously observed interaction between EBNA-LP and the retinoblastoma protein or p53.     

ZAP-70: a 70 kd protein-tyrosine kinase that associates with the TCR zeta chain.

Protein-tyrosine kinases (PTKs) play an integral role in T cell activation. Stimulation of the T cell antigen receptor (TCR) results in tyrosine phosphorylation of a number of cellular substrates. One of these is the TCR zeta chain, which can mediate the transduction of extracellular stimuli into cellular effector functions. We have recently identified a 70 kd tyrosine phosphoprotein (ZAP-70) that associates with zeta and undergoes tyrosine phosphorylation following TCR stimulation. Here we report the isolation of a cDNA clone encoding ZAP-70. ZAP-70 represents a novel PTK and is expressed in T and natural killer cells. Moreover, tyrosine phosphorylation and association of ZAP-70 with zeta require the presence of src family PTKs and provide a potential mechanism by which the src family PTKs and ZAP-70 may interact to mediate TCR signal transduction.     

Erythrocyte protein 4.1 associates with tubulin.

Protein 4.1 binds to tubulin, as determined by sedimentation and immunoelectron-microscopy analyses, at a molar ratio similar to that described for brain microtubule-associated proteins. The binding site appears to be located at the C-terminal region of tubulin. Experiments performed in situ by adding exogenous protein 4.1 to permeabilized 3T3 cells show that this protein binds to microtubule and nuclear components.     

PSL, an S phase-related p55 nuclear antigen, associates transiently with chromatin

An S phase-related nuclear 55K antigen, also designated PSL, has been characterized in various mammalian cells, using a human serum from a patient with autoimmune disorders (Barque et al., EMBO j 2 (1983) 743). In this report, we show by immunoelectron microscopy that the p55 protein associates in situ with the chromatin of rat hepatocytes. This association is a transient one, as indirect immunofluorescence studies show that PSL does not bind to individualized metaphase chromosomes. Furthermore, immunoprecipitation tests indicate that the majority of PSL is in the non-chromosomal cell fraction. These results suggest that this nuclear antigen is directly involved in the DNA replication process.