The pro-apoptotic kinase Mst1 and its caspase cleavage products are direct inhibitors of Akt1.

Akt kinases mediate cell growth and survival. Here, we report that a pro-apoptotic kinase, Mst1/STK4, is a physiological Akt1 interaction partner. Mst1 was identified as a component of an Akt1 multiprotein complex isolated from lipid raft-enriched fractions of LNCaP human prostate cancer cells. Endogenous Mst1, along with its paralog, Mst2, acted as inhibitors of endogenous Akt1. Surprisingly, mature Mst1 as well as both of its caspase cleavage products, which localize to distinct subcellular compartments and are not structurally homologous, complexed with and inhibited Akt1. cRNAs encoding full-length Mst1, and N- and C-terminal caspase Mst1 cleavage products, reverted an early lethal phenotype in zebrafish development induced by expression of membrane-targeted Akt1. Mst1 and Akt1 localized to identical subcellular sites in human prostate tumors. Mst1 levels declined with progression from clinically localized to hormone refractory disease, coinciding with an increase in Akt activation with transition from hormone naïve to hormone-resistant metastases. These results position Mst1/2 within a novel branch of the phosphoinositide 3-kinase/Akt pathway and suggest an important role in cancer progression.

Analysis of genomic differences among Clostridium botulinum type A1 strains.

BACKGROUND: Type A1 Clostridium botulinum strains are a group of Gram-positive, spore-forming anaerobic bacteria that produce a genetically, biochemically, and biophysically indistinguishable 150 kD protein that causes botulism. The genomes of three type A1 C. botulinum strains have been sequenced and show a high degree of synteny. The purpose of this study was to characterize differences among these genomes and compare these differentiating features with two additional unsequenced strains used in previous studies. RESULTS: Several strategies were deployed in this report. First, University of Massachusetts Dartmouth laboratory Hall strain (UMASS strain) neurotoxin gene was amplified by PCR and sequenced; its sequence was aligned with the published ATCC 3502 Sanger Institute Hall strain and Allergan Hall strain neurotoxin gene regions. Sequence alignment showed that there was a synonymous single nucleotide polymorphism (SNP) in the region encoding the heavy chain between Allergan strain and ATCC 3502 and UMASS strains. Second, comparative genomic hybridization (CGH) demonstrated that the UMASS strain and a strain expected to be derived from ATCC 3502 in the Centers for Disease Control and Prevention (CDC) laboratory (ATCC 3502*) differed in gene content compared to the ATCC 3502 genome sequence published by the Sanger Institute. Third, alignment of the three sequenced C. botulinum type A1 strain genomes revealed the presence of four comparable blocks. Strains ATCC 3502 and ATCC 19397 share the same genome organization, while the organization of the blocks in strain Hall were switched. Lastly, PCR was designed to identify UMASS and ATCC 3502* strain genome organizations. The PCR results indicated that UMASS strain belonged to Hall type and ATCC 3502* strain was identical to ATCC 3502 (Sanger Institute) type. CONCLUSIONS: Taken together, C. botulinum type A1 strains including Sanger Institute ATCC 3502, ATCC 3502*, ATCC 19397, Hall, Allergan, and UMASS strains demonstrate differences at the level of the neurotoxin gene sequence, in gene content, and in genome arrangement.

Zebrafish Wnt9b synteny and expression during first and second arch, heart, and pectoral fin bud morphogenesis.

Roles for Wnt9b in craniofacial development are indicated by the cleft lip mutant phenotype observed in the A/WySn mouse strain,(1) caused by a retrotransposon insertion mutation at the Wnt9b locus. Analyses of the zebrafish Wnt9b ortholog, wnt9b, were pursued to provide insight into early vertebrate craniofacial patterning events mediated by Wnt9b signaling. Zebrafish wnt9b cDNA clones were isolated and found to encode an open reading frame of 358 amino acids, with 68% amino acid identity to mouse Wnt9b and 70% amino acid identity to human WNT9B. Syntenic analyses demonstrated that wnt9b and wnt3 exist as a contiguous pair in amniote vertebrate species, and that these genes are separate in the zebrafish and Takifugu genomes. During the pharyngula period, a time of extensive growth and morphogenesis, zebrafish wnt9b exhibits discrete expression in dorsal and ventral first and second branchial arch tissues, the heart, and pectoral fin buds. These analyses suggest that in zebrafish, as in humans, wnt9b plays distinct roles in directing morphogenetic movements of developing branchial arch elements, and identify the zebrafish as a useful developmental model for the study of human craniofacial cleft lip and palate.

Caveolin-1alpha and -1beta perform nonredundant roles in early vertebrate development.

Caveolins are integral membrane proteins that localize predominantly to lipid rafts, where they oligomerize to form flask-shaped organelles termed caveolae and play important roles in membrane trafficking, signal transduction, and other cellular processes. To investigate potential roles for caveolin-1 (cav-1) in development, cav-1alpha and -1beta cDNAs were functionally characterized in the zebrafish. Cav-1alpha and -1beta mRNAs exhibited overlapping but distinct expression patterns throughout embryogenesis. Targeted depletion of either Cav-1 isoform, using antisense morpholino oligomers, resulted in a substantial loss of caveolae and dramatic neural, eye, and somite defects by 12 hours after fertilization, the time at which mRNA levels of both isoforms substantially increased in wild-type animals. Morphant phenotypes were rescued by injection of homotypic (cav-1alpha/cav-1alpha) but not heterotypic (cav-1alpha/cav-1beta) zebrafish and human cav-1 cRNAs, revealing nonredundant and evolutionarily conserved functions for the individual Cav-1 isoforms. Mutation of a known Cav-1 phosphorylation site unique to Cav-1alpha (Y14-->F) resulted in a failure to rescue the cav-1alpha morphant phenotype, verifying an essential role for Cav-1alpha specifically and implicating this residue in early developmental functions. Cav-1alpha and -1beta morphants also exhibited disruption in the actin cytoskeleton. These results support important and previously unanticipated roles for the Caveolin-1 isoforms in vertebrate organogenesis.

Partitioning of aquaporin-4 water channel mRNA and protein in gastric glands.

Immunolocalization studies in proximal, middle, and distal stomach indicated that aquaporin-4 (AQP4) protein is localized only in parietal cells located in the middle or deep regions of the gastric glands. In studies using in situ hybridization, AQP4 mRNA failed to localize in parietal cells but was identified in neighboring mucosal cells that were triangular in shape and smaller than parietal cells in size, and in columnar cells at the base of the gastric gland. This spatial separation of mRNA and protein was also observed in other species and with other kind of mRNA/protein. In neonatal and adolescent rats, the appearance of morphologically mature parietal cells was preceded by identification of mRNA-bearing triangular cells. Cells harboring both protein and mRNA were observed in postnatal rats and in the pyloric region of the glandular stomach, during induced hypergastrinaemia. The results suggest that such cells represent a transition between those that bear only mRNA and those that are terminally differentiated, expressing proteins that are related to acid secretion.

Membrane organization and function of M1 and M23 isoforms of aquaporin-4 in epithelial cells.

Aquaporin-4 (AQP4) water channels exist as heterotetramers of M1 and M23 splice variants and appear to be present in orthogonal arrays of intramembraneous particles (OAPs) visualized by freeze-fracture microscopy. We report that AQP4 forms OAPs in rat gastric parietal cells but not in parietal cells from the mouse or kangaroo rat. Furthermore, the organization of principal cell OAPs in Brattleboro rat kidney is perturbed by vasopressin (arginine vasopressin). Membranes of LLC-PK(1) cells expressing M23-AQP4 showed large, abundant OAPs, but none were detectable in cells expressing M1-AQP4. Measurements of osmotic swelling of transfected LLC-PK(1) cells using videomicroscopy, gave osmotic water permeability coefficient (P(f)) values (in cm/s) of 0.018 (M1-AQP4), 0.019 (M23-AQP4), and 0.003 (control). Quantitative immunoblot and immunofluorescence showed an eightfold greater expression of M1- over M23-AQP4 in the cell lines, suggesting that single-channel p(f) (cm(3)/s) is much greater for the M23 variant. Somatic fusion of M1- and M23-AQP4 cells (P(f) = 0.028 cm/s) yielded OAPs that were fewer and smaller than in M23 cells alone, and M1-to-M23 expression ratios ( approximately 1:4) normalized to AQP4 in M1 or M23 cells indicated a reduced single-channel p(f) for the M23 variant. Expression of an M23-AQP4-Ser(111E) mutant produced approximately 1.5-fold greater single-channel p(f) and OAPs that were up to 2.5-fold larger than wild-type M23-AQP4 OAPs, suggesting that a putative PKA phosphorylation site Ser(111) is involved in OAP formation. We conclude that the higher-order organization of AQP4 in OAPs increases single-channel osmotic water permeability by one order of magnitude and that differential cellular expression levels of the two isoforms could regulate this organization.