Previous studies from our laboratory established that large M(r) mucin glycoproteins are major apically disposed components of mouse uterine epithelial cells in vitro. The present studies demonstrate that Muc-1 represents one of the apically disposed mucin glycoproteins of mouse uterine epithelia, and that Muc-1 protein and messenger RNA (mRNA) expression are regulated in the periimplantation mouse uterus by ovarian steroids. Muc-1 expression is exclusive to the epithelial cells of the uterus under all conditions examined. Muc-1 expression is high in the proestrous and estrous stages and decreases during diestrous. Both Muc-1 protein and mRNA decline to barely detectable levels by day 4 of pregnancy, i.e. before the time of blastocyst attachment. In contrast, Muc-1 expression in the cervix and vagina is maintained during this same period. Delayed implantation was established in pregnant mice by ovariectomy and maintained by the administration of exogenous progesterone (P). Initiation of implantation was triggered by coinjection of P-maintained mice with a nidatory dose of 17 beta-estradiol (E2). Muc-1 levels in the uterine epithelia of P-maintained mice declined to low levels similar to those observed on day 4 of normal pregnancy. Coinjection of E2 did not alter Muc-1 expression, suggesting that down-regulation of Muc-1 is a P-dominated event. This was confirmed in ovariectomized nonpregnant mice, which displayed stimulation of Muc-1 expression after 6 h of E2 injection. E2-Stimulated Muc-1 expression was inhibited by the pure antiestrogen, ICI 164,384. Although P alone had no effect on Muc-1 expression, it antagonized the action of E2. Injection of pregnant mice with the antiprogestin, RU486, a known implantation inhibitor, on day 3 of pregnancy restored high level expression of Muc-1 mRNA on day 4, indicating that down-regulation of Muc-1 is P receptor mediated. Collectively, these data indicate that Muc-1 expression in mouse uterine epithelium is strongly influenced by ovarian steroids. It is suggested that the loss of Muc-1 contributes to generation of a receptive uterine state.

The protective antigen (PA) moiety of anthrax toxin transports edema factor and lethal factor to the cytosol of mammalian cells by a mechanism that depends on its ability to oligomerize and form pores in the endosomal membrane. Previously, some mutated forms of PA, designated dominant negative (DN), were found to coassemble with wild-type PA and generate defective heptameric pore-precursors (prepores). Prepores containing DN-PA are impaired in pore formation and in translocating edema factor and lethal factor across the endosomal membrane. To create a more comprehensive map of sites within PA where a single amino acid replacement can give a DN phenotype, we used automated systems to generate a Cys-replacement mutation for each of the 568 residues of PA63, the active 63-kDa proteolytic fragment of PA. Thirty-three mutations that reduced PA's ability to mediate toxicity at least 100-fold were identified in all four domains of PA63. A majority (22) were in domain 2, the pore-forming domain. Seven of the domain-2 mutations, located in or adjacent to the 2␤6 strand, the 2␤7 strand, and the 2␤10-2␤11 loop, gave the DN phenotype. This study demonstrates the feasibility of high-throughput scanning mutagenesis of a moderate sized protein. The results show that DN mutations cluster in a single domain and implicate 2␤6 and 2␤7 strands and the 2␤10-2␤11 loop in the conformational rearrangement of the prepore to the pore. They also add to the repertoire of mutations available for structure-function studies and for designing new antitoxic agents for treatment of anthrax.

We previously provided evidence that the protein encoded by the highly conserved skb1 gene is a putative regulator of Shk1, a p21Cdc42͞Rac -activated kinase (PAK) homolog in the fission yeast Schizosaccharomyces pombe. skb1 null mutants are viable and competent for mating but less elongate than wild-type S. pombe cells, whereas cells that overexpress skb1 are hyperelongated. These phenotypes suggest a possible role for Skb1 as a mitotic inhibitor. Here we show genetic interactions of both skb1 and shk1 with genes encoding key mitotic regulators in S. pombe. Our results indicate that Skb1 negatively regulates mitosis by a mechanism that is independent of the Cdc2-activating phosphatase Cdc25 but that is at least partially dependent on Shk1 and the Cdc2 inhibitory kinase Wee1. We provide biochemical evidence for association of Skb1 and Shk1 with Cdc2 in S. pombe, suggesting that Skb1 and Shk1 inhibit mitosis through interaction with the Cdc2 complex, rather than by an indirect mechanism. These results provide evidence of a previously undescribed role for PAK-related protein kinases as mitotic inhibitors. We also describe the cloning of a human homolog of skb1, SKB1Hs, and show that it can functionally replace skb1 in S. pombe. Thus, the molecular functions of Skb1-related proteins have likely been substantially conserved through evolution.

The Ste20/p21-activated kinase homolog Shk1 is essential for viability and required for normal morphology, mating, and cell cycle control in the fission yeast Schizosaccharomyces pombe. Shk1 is regulated by the p21 G protein Cdc42, which has been shown to form a complex with the SH3 domain protein Scd2 (also called Ral3). In this study, we investigated whether Scd2 plays a role in regulating Shk1 function. We found that recombinant Scd2 and Shk1 interact directly in vitro and that they interact in vivo, as determined by the two-hybrid assay and genetic analyses in fission yeast. The second of two N-terminal SH3 domains of Scd2 is both necessary and sufficient for interaction with Shk1. While full-length Scd2 interacted with only the R1 Nterminal regulatory subdomain of Shk1, a C-terminal deletion mutant of Scd2 interacted with both the R1 and R3 subdomains of Shk1, suggesting that the non-SH3 C-terminal domain of Scd2 may be involved in defining specificity in SH3 binding domain recognition. Overexpression of Scd2 stimulated the autophosphorylation activity of wild-type Shk1 in fission yeast but, consistent with results of genetic analyses, did not stimulate the activity of a Shk1 protein lacking the R1 subdomain. Results of additional two-hybrid experiments suggest that Scd2 may stimulate Shk1 catalytic function, at least in part, by positively modulating protein-protein interaction between Cdc42 and Shk1. We propose that Scd2 functions as an organizing center, or scaffold, for the Cdc42 complex in fission yeast and that it acts in concert with Cdc42 to positively regulate Shk1 function.