A cardiolipin-activated protein kinase from rat liver structurally distinct from the protein kinases C

A cardiolipin- and protease-activated protein kinase (PAK) has been isolated from cytoplasmic extracts of rat liver. The enzyme (PAK-1) phosphorylates the ribosomal protein S6-(229-239) peptide analogue and can be activated by limited proteolysis. Partial amino acid sequences of tryptic peptides derived from both the purified 116-kDa PAK-1 holoenzyme and its active catalytic fragment reveal that the catalytic domain is most related (50-58% identity) to the protein kinase C family. PAK-1 has protein and peptide substrate specificities distinct from those of known protein kinase C isoforms and is insensitive to inhibition by the protein kinase C-alpha-(19-31) pseudosubstrate peptide. Phosphatidylserine, diacylglycerol, and phorbol ester do not activate PAK-1 toward the S6 peptide substrate. However, other acidic phospholipids, the most effective being cardiolipin, activate PAK-1 to a similar extent as trypsin. The PAK-1 catalytic activities generated through activation by cardiolipin or limited proteolysis were kinetically similar, with K-m values of 3.6 and 3.4 mu M, respectively, for the S6-(229-239) peptide substrate. However, differences were observed in the catalytic activities with protamine sulfate and the glycogen synthase-(1-12) peptide analogue as substrates. It was concluded that PAK-1 is a phospholipid regulated protein kinase with a primary structure, substrate specificity, and mechanism of regulation in vitro distinct from those of any known member of the protein kinase C superfamily

Saltatory remodeling of Hox chromatin in response to rostrocaudal patterning signals

Hox genes controlling motor neuron subtype identity are expressed in rostrocaudal patterns that are spatially and temporally collinear with their chromosomal organization. Here we demonstrate that Hox chromatin is subdivided into discrete domains that are controlled by rostrocaudal patterning signals that trigger rapid, domain-wide clearance of repressive histone H3 Lys27 trimethylation (H3K27me3) polycomb modifications. Treatment of differentiating mouse neural progenitors with retinoic acid leads to activation and binding of retinoic acid receptors (RARs) to the Hox1–Hox5 chromatin domains, which is followed by a rapid domain-wide removal of H3K27me3 and acquisition of cervical spinal identity. Wnt and fibroblast growth factor (FGF) signals induce expression of the Cdx2 transcription factor that binds and clears H3K27me3 from the Hox1–Hox9 chromatin domains, leading to specification of brachial or thoracic spinal identity. We propose that rapid clearance of repressive modifications in response to transient patterning signals encodes global rostrocaudal neural identity and that maintenance of these chromatin domains ensures the transmission of positional identity to postmitotic motor neurons later in development.Leona M. and Harry B. Helmsley Charitable TrustNational Institutes of Health (U.S.) (Grant P01 NS055923)Smith Family Foundatio

Novel MicroRNA Candidates and miRNA-mRNA Pairs in Embryonic Stem (ES) Cells

MicroRNAS (miRNAS: a class of short non-coding RNAs) are emerging as important agents of post transcriptional gene regulation and integral components of gene networks. MiRNAs have been strongly linked to stem cells, which have a remarkable dual role in development. They can either continuously replenish themselves (self-renewal), or differentiate into cells that execute a limited number of specific actions (pluripotence).In order to identify novel miRNAs from narrow windows of development we carried out an in silico search for micro-conserved elements (MCE) in adult tissue progenitor transcript sequences. A plethora of previously unknown miRNA candidates were revealed including 545 small RNAs that are enriched in embryonic stem (ES) cells over adult cells. Approximately 20% of these novel candidates are down-regulated in ES (Dicer(-/-)) ES cells that are impaired in miRNA maturation. The ES-enriched miRNA candidates exhibit distinct and opposite expression trends from mmu-mirs (an abundant class in adult tissues) during retinoic acid (RA)-induced ES cell differentiation. Significant perturbation of trends is found in both miRNAs and novel candidates in ES (GCNF(-/-)) cells, which display loss of repression of pluripotence genes upon differentiation.Combining expression profile information with miRNA target prediction, we identified miRNA-mRNA pairs that correlate with ES cell pluripotence and differentiation. Perturbation of these pairs in the ES (GCNF(-/-)) mutant suggests a role for miRNAs in the core regulatory networks underlying ES cell self-renewal, pluripotence and differentiation

Evolution of marsupial and other vertebrate thyroxine-binding plasma proteins

Binding of radioactive thyroxine to proteins in the plasma of vertebrates was studied by electrophoresis followed by autoradiography. Albumin was found to be a thyroxine carrier in the blood of all studied fish, amphibians, reptiles, monotremes, marsupials, eutherians (placental mammals), and birds. Thyroxine binding to transthyretin was detected in the blood of eutherians, diprotodont marsupials, and birds, but not in blood from fish, toads, reptiles, monotremes, and Australian polyprotodont marsupials. Globulins binding thyroxine were only observed in the plasma of some mammals. Apparently, albumin is the phylogenetically oldest thyroxine carrier in vertebrate blood. Transthyretin gene expression in the liver developed in parallel, and independently, in the evolutionary lineages leading to eutherians, to diprotodont marsupials, and to birds. In contrast, high transthyretin mRNA levels, strong synthesis, and secretion of transthyretin in choroid plexus from reptiles and birds indicate that transthyretin gene expression in the choroid plexus evolved much earlier than in the liver, probably at the stage of the stem reptiles. NH-terminal sequence analysis suggests a change of transthyretin pre-mRNA splicing during evolution