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Biomedical subjects

A M Weissman

Publications and source records attributed to A M Weissman.

At least 37 records · Page 2Linked to original sources
PubMed1998

Ubiquitin and the control of protein fate in the secretory and endocytic pathways.

The modification of proteins by chains of ubiquitin has long been known to mediate targeting of cytosolic and nuclear proteins for degradation by proteasomes. In this article, we discuss recent developments that reveal the involvement of ubiquitin in the degradation of proteins retained within the endoplasmic reticulum (ER) and in the internalization of plasma membrane proteins. Both luminal and transmembrane proteins retained in the ER are now known to be retrotranslocated into the cytosol in a process that involves ER chaperones and components of the protein import machinery. Once exposed to the cytosolic milieu, retro-translocated proteins are degraded by the proteasome, in most cases following polyubiquitination. There is growing evidence that both the ubiquitin-conjugating machinery and proteasomes may be associated with the cytosolic face of the ER membrane and that they could be functionally coupled to the process of retrotranslocation. The ubiquitination of plasma membrane proteins, on the other hand, mediates internalization of the proteins, which in most cases is followed by lysosomal/vacuolar degradation. There is, however, a well-documented case of a plasma membrane protein (the c-Met receptor) for which ubiquitination results in proteasomal degradation. These recent findings imply that ubiquitin plays more diverse roles in the regulation of the fate of cellular proteins than originally anticipated.

Animals↗
PubMed1997

Serine phosphorylation-regulated ubiquitination and degradation of beta-catenin.

Several lines of evidence suggest that accumulation of cytoplasmic beta-catenin transduces an oncogenic signal. We show that beta-catenin is ubiquitinated and degraded by the proteosome and that beta-catenin stability is regulated by a diacylglycerol-independent protein kinase C-like kinase activity, which is required for beta-catenin ubiquitination. We also define a six-amino acid sequence found in both beta-catenin and the NF-kappaB regulatory protein IkappaBalpha, which, upon phosphorylation, targets both proteins for ubiquitination. Mutation of a single serine within the ubiquitination targeting sequence prevents ubiquitination of beta-catenin. Mutations within the ubiquitination targeting sequence of beta-catenin may be oncogenic.

Amino Acid Sequence↗
PubMed1997

Subcellular localization and ubiquitin-conjugating enzyme (E2) interactions of mammalian HECT family ubiquitin protein ligases.

In most instances, the transfer of ubiquitin to target proteins is catalyzed by the action of ubiquitin protein ligases (E3s). Full-length cDNAs encoding murine E6-associated protein (mE6-AP) as well as Nedd-4, a protein that is homologous to E6-AP in its C terminus, were cloned. Nedd-4 and mouse E6-AP are both enzymatically active E3s and function with members of the UbcH5 family of E2s. Mouse E6-AP, like its human counterpart, ubiquitinates p53 in the presence of human papilloma virus E6 protein, while Nedd-4 does not. Consistent with its role in p53 ubiquitination, mE6-AP was found both in the nucleus and cytosol, while Nedd-4 was found only in the cytosol. Binding studies implicate a 150-amino acid region that is 40% identical between mE6-AP and Nedd-4 as a binding site for the C-terminal portion of an E2 enzyme (UbcH5B). Nedd-4 was determined to have a second nonoverlapping E2 binding site that recognizes the first 67 amino acids of UbcH5B but not the more C-terminal portion of this E2. These findings provide the first demonstration of physical interactions between mammalian E2s and E3s and establish that these interactions occur independently of ubiquitin and an intact E3 catalytic domain. Furthermore, the presence of two E2 binding sites within Nedd-4 suggests models for ubiquitination involving multiple E2 enzymes associated with E3s.

Amino Acid Sequence↗
PubMed1997

The transmembrane domain of a carboxyl-terminal anchored protein determines localization to the endoplasmic reticulum.

UBC6 is a C-terminal membrane-anchored (type IV) protein, native to Saccharomyces cerevisiae, where it is found in the endoplasmic reticulum. When expressed in mammalian cells, this novel ubiquitin-conjugating enzyme also localizes to the endoplasmic reticulum. UBC6 lacks a lumenal domain and contains no known endoplasmic reticulum retention signals. Analysis of chimeric proteins in which the cytosolic domain of UBC is linked to a heterologous transmembrane domain, or in which the UBC6 transmembrane domain is appended to an unrelated soluble protein, led to the determination that the transmembrane domain of UBC6 plays a dominant role in its compartmental localization. The basis for the transmembrane domain-mediated subcellular targeting of UBC6 was evaluated by lengthening the wild type UBC6 hydrophobic segment from 17 to 21 amino acids, which resulted in re-targeting to the Golgi complex. A further increase in length to 26 amino acids allowed this modified protein to traverse the secretory pathway and gain expression at the plasma membrane. These findings are consistent with models in which, in the absence of dominant cytosolic or lumenal targeting determinants, proteins may be sorted within the secretory pathway based on interactions between their transmembrane domains and the surrounding lipid bilayer.

Amino Acid Sequence↗
PubMed1997

Activation of the Lck tyrosine kinase targets cell surface T cell antigen receptors for lysosomal degradation.

The mechanism by which TCR expression is regulated was explored by expressing a constitutively active form of the tyrosine kinase Lck (Lck505F) in T cells. Expression of Lck505F down-regulated TCR levels, an effect that was even more pronounced in CD45- T cells, in which the activity of this tyrosine kinase is further enhanced. Cells expressing Lck505F synthesized all TCR subunits, but lysosomal degradation of assembled receptors was enhanced. TCRs were rapidly internalized and degraded after removal of a tyrosine kinase inhibitor that had permitted cell surface expression. Finally, TCR levels on thymocytes were increased by an Lck inhibitor, and activation- but not phorbol ester-induced internalization of TCRs on Jurkat cells was prevented by inhibition or loss of Lck. These studies identify a regulated nonreceptor tyrosine kinase-mediated pathway for targeting cell surface receptors for lysosomal degradation.

Animals↗
PubMed1996

Transvaginal ultrasonography in nonpregnant and postmenopausal women.

Compared with transabdominal pelvic ultrasonography, transvaginal ultrasonography provides improved resolution for visualization of female pelvic organs, with less artifact. Transvaginal ultrasonography has superseded the transabdominal approach in many situations. Identification of uterine and adnexal masses can be facilitated with use of transvaginal ultrasonography. Transvaginal ultrasonography is also useful in the evaluation of pelvic pain and suspected pelvic inflammatory disease. Postmenopausal patients with vaginal bleeding or a palpable ovary can be triaged on the basis of transvaginal ultrasonographic findings, avoiding invasive diagnostic procedures in selected patients. Screening average-risk patients for ovarian cancer is not an accepted indication for transvaginal ultrasonography at this time.

Diagnosis, Differential↗
PubMed1996

T cell antigen receptor ubiquitination is a consequence of receptor-mediated tyrosine kinase activation.

Engagement of the T cell antigen receptor results in both its phosphorylation and its ubiquitination. T cell antigen receptor ubiquitination was evaluated in Jurkat, a well characterized human T leukemia cell line. Treatment of cells with the tyrosine kinase inhibitor herbimycin A resulted in an inhibition of receptor ubiquitination. Consistent with this, pervanadate, which increases cellular tyrosine phosphorylation, enhanced receptor ubiquitination. A requirement for receptor-mediated tyrosine kinase activity for ubiquitination was confirmed in cells lacking the tyrosine kinase p56lck and also in cells that are defective in expression of CD45, a tyrosine phosphatase that regulates the activity of p56lck. The need for tyrosine kinase activation for ubiquitination was not bypassed by directly activating protein kinase C and stimulating endocytosis of receptors. These observations establish ubiquitination of the T cell antigen receptor as a tyrosine kinase-dependent manifestation of transmembrane signaling and suggest a role for tyrosine phosphorylation in the ligand-dependent ubiquitination of mammalian transmembrane receptors.

Cells, Cultured↗
PubMed1996

Diagnostic imaging of patients with acute scrotal pain.

Common causes of acute scrotal pain include testicular torsion, epididymo-orchitis and trauma. Epididymitis in adult men is typically associated with a history of urinary tract infection or prostatitis. Testicular torsion typically presents in young adults with a sudden onset of severe scrotal pain and, frequently, a history of recurrent episodes that have spontaneously resolved. With scrotal trauma, ultrasound may demonstrate testicular fracture, hematoceles and areas of hemorrhage or testicular infarction. Since both epididymitis and testicular torsion present with scrotal pain and swelling, and may be accompanied by fever and pyuria, Doppler ultrasound or radionuclide imaging may be necessary to make the diagnosis. In acute testicular torsion, color Doppler ultrasound shows absent flow to the epididymis and testis, while nuclear imaging shows central photon-deficient areas in the ischemic hemiscrotum. In epididymo-orchitis, color Doppler ultrasound shows increased flow to the epididymis and testis, while nuclear imaging shows increased perfusion of the affected testis and hemiscrotum.

Acute Disease↗
PubMed1996

Diagnostic imaging in the evaluation of blunt abdominal trauma.

Blunt abdominal trauma is a common cause of death and nonfatal injury. Diagnostic imaging may provide useful information in hemodynamically stable patients. In addition, patients who are comatose may benefit from imaging when intra-abdominal findings would change the course of clinical management. Computed tomographic scanning of the abdomen usually provides accurate information and may allow the physician to determine whether observation or surgery is warranted.

Abdominal Injuries↗
PubMed1996

Diagnostic imaging to identify the cause of jaundice.

Imaging studies can be helpful in identifying the etiology of conjugated (direct) hyper-bilirubinemia. An elevated direct bilirubin level suggests obstructive jaundice, and ultrasound or computed tomographic (CT) imaging may identify the responsible structural lesion. Imaging can also be used to guide percutaneous biopsy. A cost-effective strategy for determining the cause of direct hyperbilirubinemia rests on ultrasound as the primary modality. Endoscopic retrograde cholangiopancreatography and CT are performed as follow-up studies only when necessary. Magnetic resonance imaging is rarely useful.

Adult↗
PubMed1995

Identification of a family of closely related human ubiquitin conjugating enzymes.

Two very closely related human E2 ubiquitin conjugating enzymes, UbfH5B and UbcH5C, have been identified. These enzymes are products of distinct genes and are 88-89% identical in amino acid sequence to the recently described human E2, UbcH5 (now designated UbcH5A), UbcH5A-C are homologous to a family of five ubiquitin conjugating enzymes from Arabidopsis thaliana, AtUBC8-12. They are also closely related to Saccharomyces cerevisiae ScUBC4 and ScUBC5, which are involved in the stress response, and play a central role in the targeting of short-lived regulatory proteins for degradation. mRNAs encoding UbcH5A-C were co-expressed in all cell lines and tissues evaluated, with UbcH5C transcripts generally expressed at the highest levels. Analysis of Southern blots suggests that there are likely to be other related members of this family. Both UbcH5B and UbcH5C form thiol ester adducts with ubiquitin, and have activities similar to UbcH5A and AtUBC8 in the conjugation of ubiquitin to target proteins in the presence of the human ubiquitin protein ligase E6-AP. These results establish the existence of a highly conserved, and widely expressed, family of human ubiquitin conjugating enzymes.

Amino Acid Sequence↗
PubMed1994

Transcriptional regulation of the T cell antigen receptor zeta subunit: identification of a tissue-restricted promoter.

The cell surface expression of T cell antigen receptors (TCR) is regulated in part by the limiting synthesis of the zeta subunit. Utilizing fragments from the 5' region of the human zeta gene, two discrete regions that promote transcription were characterized. Both of these elements are located within 125 bases of the most 3' site of transcription initiation. The more proximal (3') promoter exhibits activity in lymphoid as well as nonlymphoid cells. In contrast, the more distal (5') promoter element functions in a tissue-restricted fashion. The tissue-specific promoter is localized to a 29-base fragment. The sequence of this region is remarkable for a stretch of 11 consecutive purines that are required for activity. This element constitutes the only known tissue-specific promoter for an invariant TCR subunit. Consistent with the unique role served by the zeta subunit in assembly of the TCR, this study demonstrates that the expression of the zeta gene is regulated in a fashion distinct from other TCR components.

Base Sequence↗
PubMed1994

Activation-dependent ubiquitination of a T cell antigen receptor subunit on multiple intracellular lysines.

The T cell antigen receptor zeta chain and other T cell antigen receptor components are ubiquitinated on receptor occupancy. A systematic mutagenesis of the zeta subunit was undertaken to determine the sites of ubiquitination. Ubiquitination was found to occur in the cytoplasmic domain of zeta with multiple lysines serving as sites for mono- and polyubiquitination. The mutation of all potential sites of ubiquitination did not inhibit receptor tyrosine phosphorylation or the ubiquitination of other T cell antigen receptor subunits. Lysines introduced into nonnative positions in the zeta molecule were also able to serve as sites for ubiquitination. These findings demonstrate that once a T cell antigen receptor is targeted for ubiquitination, there is little specificity with regard to the lysine residues that are modified.

Amino Acid Sequence↗
PubMed1994

CD45 tyrosine phosphatase activity and membrane anchoring are required for T-cell antigen receptor signaling.

T cells that lack the CD45 transmembrane tyrosine phosphatase have a variety of T-cell receptor (TCR) signaling defects that are corrected by reexpression of wild-type CD45 or its intracytoplasmic domains. In this study, a chimeric molecule containing the myristylation sequence of Src and the intracellular portion of CD45, previously shown to restore function in CD45- T cells, was mutagenized to determine if membrane-associated CD45 tyrosine phosphatase activity is required to restore TCR-mediated signaling in CD45- T cells. Abolition of enzymatic activity by substitution of a serine for a critical cysteine in the first catalytic domain resulted in failure of this molecule to restore TCR signaling. Another mutation, in which a single amino acid substitution destroyed the myristylation site, resulted in failure of the chimeric molecule to partition to the plasma membrane. Although expressed at high levels and enzymatically active, this form of intracellular CD45 also failed to restore normal signaling in CD45- T cells. These findings strongly suggest that CD45's function in TCR signaling requires its proximity to membrane-associated tyrosine phosphatase substrates.

Amino Acid Sequence↗
PubMed1993

Upregulation of T cell receptor gamma chain transcription by interleukin-2.

Signals involved in TcR gene rearrangement and expression are poorly understood. The ability of interleukin 2 to control TcR gamma gene expression was examined. Precursor thymocytes grown in the presence of IL-1 and Con A develop to CD3+ T cells that express either the TcR alpha/beta or gamma/delta complex on the cell surface (1). We show here that a major subpopulation of these cells are CD4-/CD8-, gamma/delta cells expressing only low levels of TcR gamma transcripts, compared to TcR delta mRnA or to TcR gamma mRNA of gamma/delta cells grown from precursor thymocytes with IL-2 and Con A. The cells cultured with IL-1 and lectin then strongly reacted to IL-2 by upregulating the steady-state level of TcR gamma mRNA. Our findings indicate that IL-2 upregulates TcR gamma transcription by transcriptional regulatory effects in this in vitro system.

Animals↗