Iris Kemler

PUROMYCIN-SENSITIVE AMINOPEPTIDASE : SEQUENCE ANALYSIS, EXPRESSION, AND FUNCTIONAL CHARACTERIZATION

Among the molecular mechanisms that control the cell division cycle, proteolysis has emerged as a key regulatory process enabling cells to pass critical check points. Such proteolysis involves a cascade of enzymes including a multisubunit complex termed 26S proteasome. Here we report on the analysis of a novel mouse cDNA encoding the puromycin-sensitive aminopeptidase (PSA) and on its expression in COS cells and 3T3 fibroblasts. PSA is 27-40% homologous to several known Zn-binding aminopeptidases including aminopeptidase N. Immunohistochemical analysis revealed that PSA is localized to the cytoplasm and to the nucleus and associates with microtubules of the spindle apparatus during mitosis. Furthermore, puromycin and bestatin both arrested the cell cycle, leading to an accumulation of cells in G/M phase, and ultimately induced cells to undergo apoptosis at concentrations that inhibit PSA. Control experiments including cycloheximide further suggested that the induction of apoptosis by puromycin was not attributable to inhibition of protein synthesis. Taken together, these data favor the novel idea that PSA participates in proteolytic events essential for cell growth and viability.

Octamer transcription factors bind to two different sequence motifs of the immunoglobulin heavy chain promoter.

All promoters of immunoglobulin heavy chain genes contain three conserved sequence motifs: a heptamer motif CTCATGA, an octamer motif ATGCAAAT, and a TATA box. We show that, despite their different sequences, both the heptamer and the octamer motif are bound by the same octamer transcription factors (Oct factors, also referred to as OTFs), namely the lymphoid‐specific proteins Oct‐2A and Oct‐2B, as well as the ubiquitous protein Oct‐1. Even though binding to the octamer motif is stronger, a single heptamer motif can bind Oct proteins and mediate transcriptional activity in lymphoid cells. Furthermore, factor binding to the octamer motif facilitates binding to the nearby heptamer motif. We propose that the heptamer element plays a role early in B‐cell differentiation to ensure that the heavy chain promoters are transcriptionally activated before the light chain promoters, which do not contain the heptamer motif.

Octamer transcription factors and the cell type-specificity of immunoglobulin gene expression.

Antibodies are produced exclusively in B lymphocytes. The expression of the antibody‐encoding genes, the immunoglobulin (Ig) genes, is also restricted to B cells. The octamer sequence ATGCAAAT is present in the promoter and the enhancer of Ig genes, and plays an important role in its tissue‐specific expression. This sequence motif is a binding site for nuclear proteins, the so‐called octamer transcription factors (Oct or OTF factors). The Oct‐1 protein is present in all cell types analyzed so far, whereas Oct‐2A and Oct‐2B are found mainly in B lymphocytes. All three proteins show the same sequence specificity and binding affinity. It appears that the B cell‐specific expression of Ig genes is mediated at least in part by cell type‐specific Oct factors, and that there are both quantitative and qualitative differences between Oct‐1 and Oct‐2 factors. Recently, a number of other octamer factor variants were identified. Many of these may be created by alternative splicing of a primary transcript of one Oct factor gene and may serve a specific function in the fine tuning of gene expression.— Kemler, I.; Schaffner, W. Octamer transcription factors and the cell‐type specificity of immunoglobulin gene expression. FASEB J. 4: 1444‐1449; 1990.

Role of IκBα and IκBβ in the biphasic nuclear translocation of NF-κB in TNFα-stimulated astrocytes and in neuroblastoma cells

In infectious diseases of the central nervous system astrocytes respond to inflammatory cytokines like tumor necrosis factor α (TNFα) by activation of the transcription factor NF‐κB, mediated by the proteolysis of its inhibitors IκBα and IκBβ. We studied the kinetics of NF‐κB induction by TNFα in primary astrocytes, and in the neuroblastoma cell line Neuro2A, and compared it to fibroblasts. In the latter, NF‐κB DNA binding activity was induced at 30 min and remained constant up to 4 h. In contrast, in astrocytes and in Neuro2A cells NF‐κB DNA binding activity followed a biphasic pattern: it was induced after 30 min (early phase), declined after 1 h, and increased again at 2 to 4 h (late phase). The early phase was due to rapid degradation of IκBα. After 1 h IκBα was resynthesized to levels exceeding the amounts present in unstimulated cells. This paralleled the low levels of nuclear NF‐κB binding activity. The decrease was not observed when IκBα resynthesis was inhibited by cycloheximide. Degradation of both IκBα and IκBβ contributed to the late phase of induction. However, the second peak occurred also in the absence of IκBβ proteolysis, demonstrating the importance of IκBα in the formation of the biphasic nuclear translocation of NF‐κB. GLIA 26:212–220, 1999.