Schwannomas are peripheral nerve sheath tumors that often occur in the setting of an inherited tumor predisposition syndrome, including Neurofibromatosis Types 1 (NF1) and 2 (NF2), Familial Schwannomatosis (FS) and Carney Complex (CNC). Loss of the NF2 tumor suppressor (encoding NF2, or Merlin) is associated with upregulation of the Rac1 small GTPase, which is thought to play a key role in mediating tumor formation. In prior studies, we generated a mouse model of schwannomas by performing tissue-specific knockout of the CNC gene Prkar1a, which encodes the type 1A regulatory subunit of Protein Kinase A. These tumors exhibited down-regulation of Nf2 protein and an increase in activated Rac1. To assess the requirement for Rac1 in schwannoma formation, we generated a double knockout of Prkar1a and Rac1 in Schwann cells and monitored tumor formation. Loss of Rac1 reduced tumor formation by reducing proliferation and enhancing apoptosis. Surprisingly, the reduction of tumor formation was accompanied by re-expression of the Nf2 protein. Furthermore, activated Rac1 was able to downregulate Nf2 in vitro in a Pak-dependent manner. These in vivo data indicate that activation of Rac1 is responsible for suppression of Nf2 protein production; deficiency of Nf2 in Schwann cells leads to loss of cellular growth control and tumor formation.. Further, PKA activation through mutation in Prkar1a is sufficient to initiate Rac1 signaling, with subsequent reduction of Nf2 and schwannomagenesis. Although in vitro evidence has shown that loss of Nf2 activates Rac1, our data indicates that signaling between Nf2 and Rac1 occurs in a bidirectional fashion, and these interactions are modulated by PKA.
The Salmonella typhimurium pepT gene is induced nearly 30-fold in response to anaerobiosis. Anaerobic expression is dependent on the transcriptional regulator encoded by fnr (previously oxrA). Primer extension analysis and site-directed mutagenesis experiments show that pepT is transcribed from two 70 promoters. One promoter (P1) is FNR dependent and anaerobically induced, while the other (P2) appears to be constitutive. The potABCD operon is divergently transcribed from a promoter near pepT P2. Sequence analysis of pepT promoter mutations which either elevate anaerobic expression or confer constitutive expression revealed that these mutations affect the ؊10 region of the P1 or P2 promoter, respectively. The pepT200 mutation, which changes the ؊10 region of the FNR-dependent P1 promoter to the consensus, has the surprising effect of allowing five-to sevenfold anaerobic induction in the absence of FNR. We have shown that the anaerobic induction of pepT-lacZ in a pepT200 fnr strain is dependent on wild-type alleles of both crp and cya. In a pepT200 pepT-lacZ strain, -galactosidase activity was elevated aerobically in the presence of exogenous cyclic AMP (cAMP) and was elevated also in succinate minimal medium relative to its level in glucose minimal medium. Primer extension analysis confirmed that P1 is the cAMP receptor protein (CRP)-dependent promoter. Site-directed mutagenesis experiments indicated that a hybrid CRP-FNR binding site positioned at ؊41 of the P1 promoter is utilized by both FNR and CRP. CRP-cAMP also appeared to repress FNR-dependent transcription of pepT under anaerobic conditions in both the pepT ؉ and pepT200 backgrounds. Although both CRP and FNR are capable of binding the hybrid site and activating transcription of pepT, CRP requires the consensus ؊10 sequence for efficient activation.The Salmonella typhimurium pepT gene encodes an aminotripeptidase which cleaves the N-terminal amino acid from a variety of tripeptides (42). Expression of pepT is induced nearly 30-fold by growth under anaerobic conditions. Anaerobic induction requires the positive regulator encoded by the fnr (previously oxrA) gene (21, 41). FNR is a transcription factor which is required for the expression of a number of anaerobically induced genes in Escherichia coli and Salmonella (39, 41). Most FNR-dependent genes encode proteins involved in anaerobic respiratory pathways (39, 41). FNR appears to be structurally and functionally similar to the cyclic AMP (cAMP) receptor protein (CRP), a transcriptional activator which regulates transcription of target genes in response to carbon source availability (25, 39). Both FNR and CRP activate transcription by binding to well-defined palindromic DNA sequences frequently located at approximately Ϫ41 bp relative to the start of transcription. The similarity between FNR and CRP extends to their 22-bp consensus DNA binding sites, which differ at only one highly conserved position in each half site (39).Sequence analysis of the pepT promoter region (31) revealed two putative promoters ...
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