The Tpl‐2 protein serine/threonine kinase was originally identified, in a C‐terminally deleted form, as the product of an oncogene associated with the progression of Moloney murine leukemia virus‐induced T cell lymphomas in rats. The kinase domain of Tpl‐2 is homologous to the Saccharomyces cerevisiae gene product, STE11, which encodes a MAP kinase kinase kinase. This suggested that Tpl‐2 might have a similar activity. Consistent with this hypothesis, immunoprecipitated Tpl‐2 and Tpl‐2deltaC (a C‐terminally truncated mutant) phosphorylated and activated recombinant fusion proteins of the mammalian MAP kinase kinases, MEK‐1 and SEK‐1, in vitro. Furthermore, transfection of Tpl‐2 into COS‐1 cells or Jurkat T cells. markedly activated the MAP kinases, ERK‐1 and SAP kinase (JNK), which are substrates for MEK‐1 and SEK‐1, respectively. Tpl‐2, therefore, is a MAP kinase kinase kinase which can activate two MAP kinase pathways. After Raf and Mos, Tpl‐2 is the third serine/threonine oncoprotein kinase that has been shown to function as a direct activator of MEK‐1.

The PLZF (promyelocytic leukemia zinc finger) transcriptional repressor, when fused to retinoic acid receptor alpha (RAR␣), causes a refractory form of acute promyelocytic leukemia. The highly conserved N-terminal BTB (bric a brac, tramtrack, broad complex)/POZ domain of PLZF plays a critical role in this disease, since it is required for transcriptional repression by the PLZF-RAR␣ fusion protein. The crystal structure of the PLZF BTB domain revealed an obligate homodimer with a highly conserved charged pocket formed by apposition of the two monomers. An extensive structure-function analysis showed that the charged pocket motif plays a major role in transcriptional repression by PLZF. We found that mutations of the BTB domain that neutralize key charged pocket residues did not disrupt dimerization, yet abrogated the ability of PLZF to repress transcription and led to the loss of interaction with N-CoR, SMRT, and histone deacetylases (HDACs). We extended these studies to the Bcl-6 protein, which is linked to the pathogenesis of non-Hodgkin's lymphomas. In this case, neutralizing the charged pocket also resulted in loss of repression and corepressor binding. Experiments with purified protein showed that corepressor-BTB interactions were direct. A comparison of the PLZF, Bcl-6, and the FAZF (Fanconi anemia zinc finger)/ROG protein shows that variations in the BTB pocket result in differential affinity for corepressors, which predicts the potency of transcriptional repression. Thus, the BTB pocket represents a molecular structure involved in recruitment of transcriptional repression complexes to target promoters.

The promyelocytic leukemia zinc finger (PLZF) protein is a transcription factor disrupted in patients with t(11;17)(q23;q21)-associated acute promyelocytic leukemia. PLZF contains an N-terminal BTB/POZ domain which is required for dimerization, transcriptional repression, formation of high-molecular-weight DNAprotein complexes, nuclear sublocalization, and growth suppression. X-ray crystallographic data show that the PLZF BTB/POZ domain forms an obligate homodimer via an extensive interface. In addition, the dimer possesses several highly conserved features, including a charged pocket, a hydrophobic monomer core, an exposed hydrophobic surface on the floor of the dimer, and two negatively charged surface patches. To determine the role of these structures, mutational analysis of the BTB/POZ domain was performed. We found that point mutations in conserved residues that disrupt the dimer interface or the monomer core result in a misfolded nonfunctional protein. Mutation of key residues from the exposed hydrophobic surface suggests that these are also important for the stability of PLZF complexes. The integrity of the charged-pocket region was crucial for proper folding of the BTB/POZ domain. In addition, the pocket was critical for the ability of the BTB/POZ domain to repress transcription. Alteration of charged-pocket residue arginine 49 to a glutamine (mutant R49Q) yields a domain that can still dimerize but activates rather than represses transcription. In the context of full-length PLZF, a properly folded BTB/POZ domain was required for all PLZF functions. However, PLZF with the single pocket mutation R49Q repressed transcription, while the double mutant D35N/R49Q could not, despite its ability to dimerize. These results indicate that PLZF requires the BTB/POZ domain for dimerization and the charged pocket for transcriptional repression.

Studies in non-neural cells have suggested that a fall in mitochondrial membrane potential (DeltaPsiM) is one of the earliest events in apoptosis. It is not known whether neural apoptosis caused by nerve growth factor (NGF) and serum withdrawal involves a decrease in DeltaPsiM. We used epifluorescence and laser confocal microscopy with the mitochondrial potentiometric dyes chloromethyl-tetramethylrosamine methyl ester and 5,5',6, 6'-tetrachloro-1,1',3,3'-tetraethybenzimidazol carbocyanine iodide to estimate DeltaPsiM. PC12 cells were differentiated in media containing serum and NGF for 6 d before withdrawal of trophic support. After washing, the cells were incubated with media containing serum and NGF (M/S+N), media without serum and NGF, or media with the "trophic-like" monoamine oxidase B inhibitor, (-)-deprenyl. Mitochondria in cells without trophic support underwent a progressive shift to lower DeltaPsiM values that was significant by 3 hr after washing. The percentages of cells with nuclear chromatin condensation or nuclear DNA fragmentation were not significantly increased above those for cells in M/S+N until 6 hr after washing. Replacement of cells into M/S+N or treatment with (-)-deprenyl markedly reduced the proportion of mitochondria with decreased DeltaPsiM. Measurements of cytoplasmic peroxyl radical levels with 2',7'-dihydrodichlorofluorescein fluorescence and intramitochondrial Ca2+ with dihydro-rhodamine-2-acetylmethyl ester indicated that cytoplasmic peroxyl radical levels were not increased until after 6 hr, whereas increases in intramitochondrial Ca2+ paralleled the decreases in DeltaPsiM. (-)-Deprenyl appeared to alter the relationship between intramitochondrial Ca2+ levels and DeltaPsiM, possibly through its reported capacity to increase the synthesis of proteins such as BCL-2.

Cystic fibrosis is a fatal genetic disease, most frequently caused by the retention of the CFTR (cystic fibrosis transmembrane conductance regulator) mutant protein in the endoplasmic reticulum (ER). The binding of the 14-3-3 protein to the CFTR regulatory (R) domain has been found to enhance CFTR trafficking to the plasma membrane. To define the mechanism of action of this protein–protein interaction, we have examined the interaction in vitro. The disordered multiphosphorylated R domain contains nine different 14-3-3 binding motifs. Furthermore, the 14-3-3 protein forms a dimer containing two amphipathic grooves that can potentially bind these phosphorylated motifs. This results in a number of possible binding mechanisms between these two proteins. Using multiple biochemical assays and crystal structures, we show that the interaction between them is governed by two binding sites: The key binding site of CFTR (pS768) occupies one groove of the 14-3-3 dimer, and a weaker, secondary binding site occupies the other binding groove. We show that fusicoccin-A, a natural-product tool compound used in studies of 14-3-3 biology, can stabilize the interaction between 14-3-3 and CFTR by selectively interacting with a secondary binding motif of CFTR (pS753). The stabilization of this interaction stimulates the trafficking of mutant CFTR to the plasma membrane. This definition of the druggability of the 14-3-3–CFTR interface might offer an approach for cystic fibrosis therapeutics.