Multiple ETS family proteins regulate PF4 gene expression by binding to the same ETS binding site

In previous studies on the mechanism underlying megakaryocyte-specific gene expression, several ETS motifs were found in each megakaryocyte-specific gene promoter. Although these studies suggested that several ETS family proteins regulate megakaryocyte-specific gene expression, only a few ETS family proteins have been identified. Platelet factor 4 (PF4) is a megakaryocyte-specific gene and its promoter includes multiple ETS motifs. We had previously shown that ETS-1 binds to an ETS motif in the PF4 promoter. However, the functions of the other ETS motifs are still unclear. The goal of this study was to investigate a novel functional ETS motif in the PF4 promoter and identify proteins binding to the motif. In electrophoretic mobility shift assays and a chromatin immunoprecipitation assay, FLI-1, ELF-1, and GABP bound to the -51 ETS site. Expression of FLI-1, ELF-1, and GABP activated the PF4 promoter in HepG2 cells. Mutation of a -51 ETS site attenuated FLI-1-, ELF-1-, and GABP-mediated transactivation of the promoter. siRNA analysis demonstrated that FLI-1, ELF-1, and GABP regulate PF4 gene expression in HEL cells. Among these three proteins, only FLI-1 synergistically activated the promoter with GATA-1. In addition, only FLI-1 expression was increased during megakaryocytic differentiation. Finally, the importance of the -51 ETS site for the activation of the PF4 promoter during physiological megakaryocytic differentiation was confirmed by a novel reporter gene assay using in vitro ES cell differentiation system. Together, these data suggest that FLI-1, ELF-1, and GABP regulate PF4 gene expression through the -51 ETS site in megakaryocytes and implicate the differentiation stage-specific regulation of PF4 gene expression by multiple ETS factors.     

Investigation of the stability of yeast rad52 mutant proteins uncovers post-translational and transcriptional regulation of Rad52p

We investigated the stability of the Saccharomyces cerevisiae Rad52 protein to learn how a cell controls its quantity and longevity. We measured the cellular levels of wild-type and mutant forms of Rad52p when expressed from the RAD52 promoter and the half-lives of the various forms of Rad52p when expressed from the GAL1 promoter. The wild-type protein has a half-life of 15 min. rad52 mutations variably affect the cellular levels of the protein products, and these levels correlate with the measured half-lives. While missense mutations in the N terminus of the protein drastically reduce the cellular levels of the mutant proteins, two mutations--one a deletion of amino acids 210-327 and the other a missense mutation of residue 235--increase the cellular level and half-life more than twofold. These results suggest that Rad52p is subject to post-translational regulation. Proteasomal mutations have no effect on Rad52p half-life but increase the amount of RAD52 message. In contrast to Rad52p, the half-life of Rad51p is >2 hr, and RAD51 expression is unaffected by proteasomal mutations. These differences between Rad52p and Rad51p suggest differential regulation of two proteins that interact in recombinational repair.     

A short-lived nuclear phosphoprotein encoded by the human ets-2 proto-oncogene is stabilized by activation of protein kinase C.

The human ets-2 gene is a homolog of the v-ets oncogene of the E26 virus and codes for a 56-kilodalton nuclear protein. The ets-2 protein is phosphorylated and has a rapid turnover, with a half-life of 20 min. When human lymphocytic CEM cells were treated with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), the level of the ets-2 protein was quickly elevated 5- to 20-fold. This effect of TPA was mimicked by a synthetic diacylglycerol, 1-oleoyl-2-acetyl glycerol, and was blocked by the protein kinase C inhibitor H7, indicating that protein kinase C is involved in the induction. The increase in the ets-2 protein was due to stabilization of the protein, because the protein had a half-life of more than 2 h in the presence of TPA and the ets-2 mRNA level did not increase significantly upon TPA treatment. The protein synthesis inhibitor cycloheximide enhanced the effect of TPA on the ets-2 protein and could itself slow turnover of the protein. Properties of the ets-2 protein, such as nuclear localization, phosphorylation, rapid turnover, and response to protein kinase C, indicate that this protein belongs to a group of oncogene proteins which are generally thought to have regulatory functions in the nucleus (e.g., myc, fos, myb, and p53). Our results suggest that protein kinase C, either directly or indirectly, regulates the level of the ets-2 protein by posttranslational mechanisms.     

Stimulation of protein synthesis in COS cells transfected with variants of the alpha-subunit of initiation factor eIF-2.

The role of eukaryotic initiation factor 2 (eIF-2) phosphorylation in translational control has been demonstrated in vivo by overexpressing variant forms of eIF-2 alpha that are not phosphorylated. COS-1 cells transiently transfected with expression vectors for human eIF-2 alpha contain 10-20-fold more eIF-2 alpha subunit than the endogenous COS cell eIF-2 trimeric complex. Expression of the variant form of eIF-2 alpha, Ser51Asp, where Asp replaces Ser51, causes inhibition of protein synthesis, whereas the Ser48Asp variant does not. When either Ser48 or Ser51 is replaced by Ala, the variants stimulate dihydrofolate reductase synthesis when the eIF-2 alpha kinase, DAI, is activated. In order to elucidate these mechanisms, we have separated eIF-2 trimeric complexes from free overexpressed eIF-2 alpha subunits by fast protein liquid chromatography Superose chromatography. Pulse-labeled cells transfected with wild-type or variant DNAs produced eIF-2 preparations with greater than 10-fold higher specific radioactivity in the alpha-subunit compared to the gamma-subunit, thus demonstrating that the human eIF-2 alpha produced from the plasmids readily exchanges into COS cell eIF-2 complexes. Both wild-type and Ser48Ala variant forms of the free 2 alpha-subunit, further purified by MonoQ chromatography, are poor substrates for the heme-regulated eIF-2 alpha kinase, HRI, but are good substrates for double-stranded RNA-activated inhibitor in vitro; the Ser51Ala variant subunit is not phosphorylated by either kinase. None of the purified free eIF-2 alpha subunits inhibits phosphorylation of eIF-2 in vitro, even at up to 8-fold molar excess. Examination of the extent of eIF-2 alpha phosphorylation in the COS cell eIF-2 complexes by two-dimensional polyacrylamide gel electrophoresis shows that the stimulation of dihydrofolate reductase synthesis by the Ser51Ala variant is most readily explained by failure of eIF-2 to be phosphorylated. Stimulation by the Ser48Ala variant appears to occur by mitigation of the effect of phosphorylation at Ser51 since the double variant, Ser48Ala-Ser51Asp, inhibits protein synthesis less than the single variant Ser51Asp. The evidence argues strongly against there being a second site of phosphorylation involved in translational repression.     

In vitro evolution of thermostable p53 variants

The tumor suppressor p53 is conformationally unstable at physiological temperature. Even the activated p53delta30 variant, which lacks the self-inhibiting carboxy terminal domain, has a half-life of only 8 min at 37 degrees C in vitro. We have developed a genetic approach to identify p53 variants that stabilize the active conformation. The human p53delta30 gene was randomly mutated, and the resulting library was expressed in Escherichia coli under conditions that apparently denatured the parental protein. Stable p53 variants were identified based on their ability to specifically bind a p53 consensus site. The initial thermostable variants were randomly recombined by DNA shuffling, and substitutions that were functionally additive or synergistic were identified in a second more stringent round of screening. The DNA binding activity of N239Y/N268D/E336V p53delta30 variant has a half-life of 100 min at 37 degrees C, 12 times longer than that of the parental protein. The thermostable variants should be more amenable to crystallographic studies and more effective in gene therapies than the wild-type protein.     

Differential processing and turnover of the oncogenically activated neu/erb B2 gene product and its normal cellular counterpart

In nontransformed DHFR/G-8 cells (NIH 3T3 cells transfected with normal rat neu gene), the normal neu gene product was initially synthesized as a 170-kDa protein bearing endoglycosidase H-sensitive oligosaccharide chains and was then processed to a 175-kDa mature form with endoglycosidase H-resistant, endoglycosidase F-sensitive oligosaccharide chains. Most of this 175-kDa mature form appeared on the cell surface 2 h following synthesis and showed a half-life of approximately 3 h. In the presence of a growth factor(s) partially purified from bovine kidney, the half-life of this 175-kDa normal neu gene product was shortened to less than 30 min. In B104-1-1 cells (NIH 3T3 cells transfected with neu gene activated oncogenically by a point mutation that changes a valine residue to a glutamic acid residue in the putative transmembrane region), the oncogenically activated neu gene product was also synthesized as a 170-kDa precursor with endoglycosidase H-sensitive oligosaccharide chains. However, this 170-kDa precursor diminished very fast and was only partially processed to a 185-kDa mature form which exhibited a half-life of less than 30 min. The 185-kDa activated neu gene product possessed an unidentified post-translational modification in addition to N-linked oligosaccharide chains. Both the precursor and mature forms of the mutationally activated neu gene product showed increased tyrosine-specific phosphorylation as compared with those of their normal counterparts in DHFR/G-8 cells. The mutationally activated neu gene product in B104-1-1 cells shared several features which have been reported previously for the ligand-activated platelet-derived growth factor receptor in v-sis- or c-sis-transformed cells. These properties include: 1) accelerated turnover of the precursor and mature forms compared with the rates of turnover of its normal counterparts, 2) insensitivity of this rapid turnover to lysosomotropic amines, and 3) increased in vivo tyrosine-specific phosphorylation of both the precursor and mature forms. These findings suggest that the mutationally activated neu gene product may transform the cells by mimicking ligand-induced activation.     

Serine 123 Phosphorylation Modulates p21 Protein Stability and Activity by Suppressing Ubiquitin-independent Proteasomal Degradation

The cyclin-dependent kinase inhibitor p21^Waf1/Cip1 is a major regulator of the cell cycle and plays an important role in many cellular processes, including differentiation, stress response, apoptosis, and tumorigenesis. We previously cloned the gene encoding dog p21 and found that unlike its human ortholog, dog p21 is expressed as two isoforms, one high molecular mass band of 23 kDa and one low molecular mass band of 19 kDa. In the current study, we found that the high molecular mass band is phosphorylated, whereas the low molecular mass band is hypophosphorylated. Moreover, by generating multiple mutants of dog p21, we found that serine 123 and proline 124, which form a consensus site for proline-directed phosphorylation, are required for expression of the high molecular mass p21 isoform through phosphorylation at serine 123. Most importantly, we showed that serine 123 phosphorylation inhibits ubiquitin-independent proteasomal degradation of p21 protein and subsequently, prolongs p21 protein half-life and enhances the ability of p21 to suppress cell proliferation. Taken together, these data reveal that serine 123 phosphorylation modulates p21 protein stability and activity by suppressing ubiquitin-independent proteasomal degradation.