Nucleocytoplasmic shuttling of Bruton's tyrosine kinase.
(73/21932)
Bruton's tyrosine kinase (Btk), a nonreceptor cytoplasmic tyrosine kinase belonging to the Tec family of kinases, has been shown to be critical for B cell proliferation, differentiation, and signaling. Loss-of-function mutations in the Btk gene lead to X-linked agammaglobulinemia (XLA), a primary immunodeficiency in humans, and the less severe condition xid in mice. Although Btk is mainly localized in the cytoplasm under steady state conditions, it translocates to the plasma membrane upon growth factor stimulation and cross-linking of the B cell receptor. Nevertheless, in ectopically as well as endogenously Btk-expressing cells, it can also translocate to the nucleus. Deletion of the pleckstrin homology (PH) domain (DeltaPH1) leads, however, to an even redistribution of Btk within the nucleus and cytoplasm in the majority of transfected cells. In contrast, an SH3-deleted (DeltaSH3) mutant of Btk has been found to be predominantly nuclear. We also demonstrate that the nuclear accumulation of DeltaPH1 is dependent on Src expression. This nucleocytoplasmic shuttling is sensitive to the exportin 1/CRM1-inactivating drug, leptomycin B, indicating that Btk utilizes functional nuclear export signals. In addition, while the DeltaPH1 mutant of Btk was found to be active and tyrosine-phosphorylated in vivo, DeltaSH3 displayed decreased autokinase activity and was not phosphorylated. Our findings indicate that the nucleocytoplasmic shuttling of Btk has implications regarding potential targets inside the nucleus, which may be critical in gene regulation during B cell development and differentiation. (+info)
The cytoplasmic domain of the interleukin-6 receptor gp80 mediates its basolateral sorting in polarized madin-darby canine kidney cells.
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The IL-6 receptor complex is expressed in different polarized epithelial cells such as liver hepatocytes and intestinal cells. It consists of two subunits: gp80, which binds the ligand, and gp130, which is responsible for signal transduction. In stably transfected Madin-Darby canine kidney (MDCK) cells we have studied the localization of the human IL-6 receptor subunits and found that gp80 and gp130 are predominantly expressed at the basolateral membrane. Analysis of MDCK cells expressing truncated forms of gp80 or gp130 showed that loss of the cytoplasmic domains results in apical delivery. Expression of deletion mutants of gp80 in MDCK cells led to the identification of two discontinous motifs responsible for basolateral sorting: a membrane-proximal tyrosine-based motif (YSLG) and a more membrane-distal dileucine-type motif (LI). Activation of signal transducer and activator of transcription-3 (STAT-3) only occurred via basolaterally located gp80, suggesting that endogenous gp130 is also constrained to the basolateral plasma membrane. Our identification of a basolateral sorting signal within the cytoplasmic region of gp80 for the first time attributes a function to this domain. (+info)
The p58-positive pre-golgi intermediates consist of distinct subpopulations of particles that show differential binding of COPI and COPII coats and contain vacuolar H(+)-ATPase.
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We have studied the structural and functional properties of the pre-Golgi intermediate compartment (IC) in normal rat kidney cells using analytical cell fractionation with p58 as the principal marker. The sedimentation profile (sediterm) of p58, obtained by analytical differential centrifugation, revealed in steady-state cells the presence of two main populations of IC elements whose average sedimentation coefficients, s(H)=1150+/-58S ('heavy') and s(L)=158+/-8S ('light'), differed from the s-values obtained for elements of the rough and smooth endoplasmic reticulum. High resolution analysis of these subpopulations in equilibrium density gradients further revealed that the large difference in their s-values was mainly due to particle size. The 'light' particle population contained the bulk of COPI and COPII coats, and redistribution of p58 to these particles was observed in transport-arrested cells, showing that the two types of elements are also compositionally distinct and have functional counterparts in intact cells. Using a specific antibody against the 16 kDa proteolipid subunit of the vacuolar H(+)-ATPase, an enrichment of the V(o )domain of the ATPase was observed in the p58-positive IC elements. Interestingly, these elements could contain both COPI and COPII coats and their density distribution was markedly affected by GTP(&ggr;)S. Together with morphological observations, these results demonstrate that, in addition to clusters of small tubules and vesicles, the IC also consists of large-sized structures and corroborate the proposal that the IC elements contain an active vacuolar H(+)-ATPase. (+info)
Protein ligands to HuR modulate its interaction with target mRNAs in vivo.
(76/21932)
AU-rich elements (AREs) present in the 3' untranslated regions of many protooncogene, cytokine, and lymphokine messages target them for rapid degradation. HuR, a ubiquitously expressed member of the ELAV (embryonic lethal abnormal vision) family of RNA binding proteins, selectively binds AREs and stabilizes ARE-containing mRNAs in transiently transfected cells. Here, we identify four mammalian proteins that bind regions of HuR known to be essential for its ability to shuttle between the nucleus and the cytoplasm and to stabilize mRNA: SETalpha, SETbeta, pp32, and acidic protein rich in leucine (APRIL). Three have been reported to be protein phosphatase 2A inhibitors. All four ligands contain long, acidic COOH-terminal tails, while pp32 and APRIL share a second motif: rev-like leucine-rich repeats in their NH(2)-terminal regions. We show that pp32 and APRIL are nucleocytoplasmic shuttling proteins that interact with the nuclear export factor CRM1 (chromosomal region maintenance protein 1). The inhibition of CRM1 by leptomycin B leads to the nuclear retention of pp32 and APRIL, their increased association with HuR, and an increase in HuR's association with nuclear poly(A)+ RNA. Furthermore, transcripts from the ARE-containing c-fos gene are selectively retained in the nucleus, while the cytoplasmic distribution of total poly(A)+ RNA is not altered. These data provide evidence that interaction of its ligands with HuR modulate HuR's ability to bind its target mRNAs in vivo and suggest that CRM1 is instrumental in the export of at least some cellular mRNAs under certain conditions. We discuss the possible role of these ligands upstream of HuR in pathways that govern the stability of ARE-containing mRNAs. (+info)
Rapid transport of internalized P-selectin to late endosomes and the TGN: roles in regulating cell surface expression and recycling to secretory granules.
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Prior studies on receptor recycling through late endosomes and the TGN have suggested that such traffic may be largely limited to specialized proteins that reside in these organelles. We present evidence that efficient recycling along this pathway is functionally important for nonresident proteins. P-selectin, a transmembrane cell adhesion protein involved in inflammation, is sorted from recycling cell surface receptors (e.g., low density lipoprotein [LDL] receptor) in endosomes, and is transported from the cell surface to the TGN with a half-time of 20-25 min, six to seven times faster than LDL receptor. Native P-selectin colocalizes with LDL, which is efficiently transported to lysosomes, for 20 min after internalization, but a deletion mutant deficient in endosomal sorting activity rapidly separates from the LDL pathway. Thus, P-selectin is sorted from LDL receptor in early endosomes, driving P-selectin rapidly into late endosomes. P-selectin then recycles to the TGN as efficiently as other receptors. Thus, the primary effect of early endosomal sorting of P-selectin is its rapid delivery to the TGN, with rapid turnover in lysosomes a secondary effect of frequent passage through late endosomes. This endosomal sorting event provides a mechanism for efficiently recycling secretory granule membrane proteins and, more generally, for downregulating cell surface receptors. (+info)
Spontaneous release of cytosolic proteins from posttranslational substrates before their transport into the endoplasmic reticulum.
(78/21932)
In posttranslational translocation in yeast, completed protein substrates are transported across the endoplasmic reticulum membrane through a translocation channel formed by the Sec complex. We have used photo-cross-linking to investigate interactions of cytosolic proteins with a substrate synthesized in a reticulocyte lysate system, before its posttranslational translocation through the channel in the yeast membrane. Upon termination of translation, the signal recognition particle (SRP) and the nascent polypeptide-associated complex (NAC) are released from the polypeptide chain, and the full-length substrate interacts with several different cytosolic proteins. At least two distinct complexes exist that contain among other proteins either 70-kD heat shock protein (Hsp70) or tailless complex polypeptide 1 (TCP1) ring complex/chaperonin containing TCP1 (TRiC/CCT), which keep the substrate competent for translocation. None of the cytosolic factors appear to interact specifically with the signal sequence. Dissociation of the cytosolic proteins from the substrate is accelerated to the same extent by the Sec complex and an unspecific GroEL trap, indicating that release occurs spontaneously without the Sec complex playing an active role. Once bound to the Sec complex, the substrate is stripped of all cytosolic proteins, allowing it to subsequently be transported through the membrane channel without the interference of cytosolic binding partners. (+info)
Neuronal nitric oxide synthase localizes through multiple structural motifs to the sarcolemma in mouse myotubes.
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In skeletal muscle, neuronal nitric oxide synthase is localized at the sarcolemma in association with the dystrophin glycoprotein complex (DGC). The nNOS N-terminal 231 amino acids comprise a PDZ domain (residues 1-100) and a beta-hairpin finger loop (residues 101-130) which binds alpha-syntrophin located in the DGC. Endogenous nNOS and GFP-tagged nNOS localize to the sarcolemma in mouse C2C12 myotubes. Expression of GFP-tagged nNOS domains in C2C12 myotubes reveals that the PDZ domain and the beta-hairpin finger loop of nNOS are independently capable of localizing to the sarcolemma of C2C12 myotubes. Binding studies indicate that alpha-syntrophin binds only to the beta-hairpin finger loop and not the PDZ domain of nNOS. nNOS may bind to proteins in addition to alpha-syntrophin at muscle sarcolemma. (+info)
Permissive role of protein kinase C alpha but not protein kinase C delta in sphingosine 1-phosphate-induced Rho A activation in C2C12 myoblasts.
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Rho GTPases participate in various important signaling pathways and have been implicated in myogenic differentiation. Here the first evidence is provided that in C2C12 myoblasts sphingosine 1-phosphate (SPP) rapidly and transiently induced membrane association of Rho A in a pertussis toxin-insensitive manner. The bioactive lipid preferentially relocalized the GTPase to Golgi-enriched membrane. Translocation of Rho A was abolished by inhibition or down-regulation of protein kinase C (PKC). Notably, treatment with Go6976, an inhibitor of conventional PKCs, which selectively blocked PKC alpha in these cells, prevented SPP-induced Rho A translocation. Conversely rottlerin, a selective inhibitor of PKC delta, was without effect, demonstrating that SPP signaling to Rho A involves PKC alpha but not PKC delta activation. This novel functional relationship between the two proteins may have a role in SPP-mediated regulation of downstream effectors. (+info)