The Aspergillus nidulans transcription factor PacC, which mediates pH regulation, is proteolytically processed to a functional form in response to ambient alkaline pH. The full-length PacC form is unstable in the presence of an operational pH signal transduction pathway, due to processing to the relatively stable short functional form. We have characterized and used an extensive collection of pacC mutations, including a novel class of "neutrality-mimicking" pacC mutations having aspects of both acidity-and alkalinity-mimicking phenotypes, to investigate a number of important features of PacC processing. Analysis of mutant proteins lacking the major translation initiation residue or truncated at various distances from the C terminus showed that PacC processing does not remove N-terminal residues, indicated that processing yields slightly heterogeneous products, and delimited the most upstream processing site to residues ϳ252 to 254. Faithful processing of three mutant proteins having deletions of a region including the predicted processing site(s) and of a fourth having 55 frameshifted residues following residue 238 indicated that specificity determinants reside at sequences or structural features located upstream of residue 235. Thus, the PacC protease cuts a peptide bond(s) remote from these determinants, possibly thereby resembling type I endonucleases. Downstream of the cleavage site, residues 407 to 678 are not essential for processing, but truncation at or before residue 333 largely prevents it. Ambient pH apparently regulates the accessibility of PacC to proteolytic processing. Alkalinity-mimicking mutations L259R, L266F, and L340S favor the protease-accessible conformation, whereas a protein with residues 465 to 540 deleted retains a protease-inaccessible conformation, leading to acidity mimicry. Finally, not only does processing constitute a crucial form of modulation for PacC, but there is evidence for its conservation during fungal evolution. Transgenic expression of a truncated PacC protein, which was processed in a pH-independent manner, showed that appropriate processing can occur in Saccharomyces cerevisiae.A growing class of transcription factors is activated by the proteolytic removal of protein domains which negatively modulate their activity. These negatively acting domains can be provided in trans (i.e., by another protein in a complex) or in cis (i.e., by a region within the transcription factor's primary translation product). Examples of the former are the p50-p52 NF-B family and their negative regulators, the IB proteins, which regulate human genes involved in immune and inflammatory responses (reviewed in reference 37), and their respective Drosophila homologues dorsal and cactus (reviewed in reference 3), which establish the dorsal-ventral polarity of the fly embryo and mediate the Drosophila immune response (19). Examples of the latter include the p105 precursor of NF-B p50 (whose C-terminal moiety is homologous to a trans-acting member of the IB family); the sterol regulatory element bi...

Snail1 is a central regulator of epithelial cell adhesion and movement in epithelial-to-mesenchymal transitions (EMTs) during embryo development; a process reactivated during cancer metastasis. While induction of Snail1 transcription precedes EMT induction, post-translational regulation of Snail1 is also critical for determining Snail1's protein level, subcellular localization, and capacity to induce EMT. To identify novel post-translational regulators of Snail1, we developed a live cell, bioluminescence-based screen. From a human kinome RNAi screen, we have identified Lats2 kinase as a novel regulator of Snail1 protein level, subcellular localization, and thus, activity. We show that Lats2 interacts with Snail1 and directly phosphorylates Snail1 at residue T203. This occurs in the nucleus and serves to retain Snail1 in the nucleus thereby enhancing its stability. Lats2 was found to positively influence cellular EMT and tumour cell invasion, in a Snail1-dependent manner. Indeed during TGFb-induced EMT Lats2 is activated and Snail1 phosphorylated at T203. Analysis in mouse and zebrafish embryo development confirms that Lats2 acts as a positive modulator of Snail1 protein level and potentiates its in vivo EMT activity.

The Aspergillus nidulans zinc finger transcription factor PacC is activated by proteolytic processing in response to ambient alkaline pH. The pH-regulated step is the transition of full-length PacC from a closed to an open, protease-accessible conformation. Here we show that in the absence of ambient pH signaling, the C-terminal negative-acting domain prevents the nuclear localization of full-length closed PacC. In contrast, the processed PacC form is almost exclusively nuclear at any ambient pH. In the presence of ambient pH signaling, the fraction of PacC that is in the open conformation but has not yet been processed localizes to the nucleus. Therefore, ambient alkaline pH leads to an increase in nuclear PacC by promoting the proteolytic elimination of the negative-acting domain to yield the processed form and by increasing the proportion of full-length protein that is in the open conformation. These findings explain why mutations resulting in commitment of PacC to processing irrespective of ambient pH lead to permanent PacC activation and alkalinity mimicry. A nuclear import signal that targets Escherichia coli ␤-galactosidase to the nucleus has been located to the PacC zinc finger region. A mutation abolishing DNA binding does not prevent nuclear localization of the processed form, showing that PacC processing does not lead to nuclear localization by passive diffusion of the protein made possible by the reduction in size, followed by retention in the nucleus after DNA binding.Proteolytic processing activation of transcription factors in response to their cognate environmental signals occurs across distant groups of eukaryotic organisms. pH regulation of gene expression in the mold Aspergillus nidulans is one such example. Here, the key regulatory zinc finger protein PacC activates alkaline genes and represses acidic genes according to the needs imposed by ambient pH, thereby providing the organism with one prerequisite for growing in environments as acidic as pH 2.5 or as alkaline as pH 10.5 (7, 59). Other prototypical members of the group of transcription factors activated by proteolytic processing are the immune and inflammatory response regulator NF-B (23, 58), the Drosophila melanogaster cubitus interruptus (Ci) zinc finger factor (the transducer of the hedgehog signal) (29, 53), and the sterol regulatory elementbinding protein (SREBP), which switches on genes for cholesterol biosynthesis and fat metabolism (5, 6).The zinc finger transcription factor PacC is synthesized as a 674-residue precursor. At alkaline ambient pH, a signal transmitted to PacC by the orphan pal gene signal transduction pathway (13,14,37,43,44) results in a conformational change leading to an open conformation in which PacC is accessible to a processing protease (18,41,47). This protease removes ϳ400 residues from the C terminus, which includes a negative-acting domain. The resulting product (248 to 250 residues) (41) is fully competent in transcriptional regulation through 5Ј-GCC ARG-3Ј sites (20) in the promoters of both alkaline...

Snail proteins are C2H2 class zinc finger transcription factors involved in different processes during embryonic development, as well as in several adult pathologies including cancer and organ fibrosis. The expression of Snail transcription factors is tightly regulated at the transcriptional level and their activity is modulated by their subcellular localization. Given the importance of this gene family in physiology and pathology, it is essential to understand the mechanisms by which Snail proteins are imported into or exported out of the nucleus. Here we show that several importins mediate the nuclear import of the human Snail proteins and we identify a unique nuclear localization signal (NLS), recognized by all the importins, that has been conserved during the evolution of the Snail family. This NLS is characterized by the presence of basic residues at defined positions in at least three consecutive zinc fingers. Interestingly, the consensus residues for importin-binding are also involved in DNA binding, suggesting that importins could prevent non-specific binding of these transcription factors to cytoplasmic polyanions. Importantly, the identified basic residues are also conserved in other families of C2H2 transcription factors whose nuclear localization requires the zinc finger region.

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