p21-activated kinase 1 interacts with and phosphorylates histone H3 in breast cancer cells

Stimulation of p21-activated kinase-1 (Pak1) signaling promotes motility, invasiveness, anchorage-independent growth and abnormal mitotic assembly in human breast cancer cells. Here, we provide new evidence that, before the onset of mitosis, activated Pak1 is specifically localized with the chromosomes during prophase and on the centrosomes in metaphase and moves to the contraction ring during cytokinesis. To identify mitosis-specific substrates of Pak1, we screened a synchronized G₂–M expression library by using a glutathione transferase Pak1 solid-phase-based kinase reaction. This analysis identified histone H3 as a substrate of Pak1 both in vitro and in vivo, and it specifically interacted with Pak1 but not Pak2 or Pak3. Site-directed mutagenesis indicated that Pak1 phosphorylates histone H3 on Ser10. Expressions of the wild-type, or catalytically active, Pak1 caused it to appear at the poles corresponding to mitotic centrosomes in a variety of mammalian cells. Together, these results suggest for the first time that Pak1 interacts with and phosphorylates histone H3 and may thus influence the Pak1–histone H3 pathway, which in turn may influence mitotic events in breast cancer cells.     

Divergence of mitochondrial dna is not corroborated by nuclear dna, morphology, or behavior in Drosophila simulans

We ask whether the observed mitochondrial DNA (mtDNA) population subdivision of Drosophila simulans is indicative of organismal structure or of specific processes acting on the mitochondrial genome. Factors either intrinsic or extrinsic to the host genome may influence the evolutionary dynamics of mtDNA. Potential intrinsic factors include adaptation of the mitochondrial genome and of nucleomitochondrial gene complexes specific to the local environment. An extrinsic force that has been shown to influence mtDNA evolution in invertebrates is the bacterial endosymbiont Wolbachia. Evidence presented in this study suggests that mtDNA is not a good indicator of organismal subdivision in D. simulans. Furthermore, there is no evidence to suggest that Wolbachia causes any reduction in nuclear gene flow in this species. The observed differentiation in mtDNA is not corroborated by data from NADH: ubiquinone reductase 75kD subunit precursor or the Alcohol dehydrogenase-related loci, from the shape or size of the male genital arch, or from assortative premating behavior. We discuss these results in relation to a mitochondrial genetic species concept and the potential for Wolbachia-induced incompatibility to be a mechanism of speciation in insects. We conclude with an iterated appeal to include phylogenetic and statistical tests of neutrality as a supplement to phylogenetic and population genetic analyses when using mtDNA as an evolutionary marker.     

Do amyloids remember their origin? New insights into the prion species barrier

Excellent work demonstrates that, in the test tube, prion protein itself is responsible for its species specificity, although questions remain about whether this occurs in the same way in the organism.     

Chemically induced supernumerary lumbar ribs in CD-1 mice: size distribution and dose response

Supernumerary ribs (SNR) of differing sizes are commonly observed in rodent developmental toxicity studies, and the significance of treatment-related increases in SNR in standard studies has been contentious. We induced dose-related increases in SNR in fetal CD-1 mice by treating on gestation days 7-8 with benomyl (BEN; 0, 75, 150 mg/kg/d), dinoseb (DIN; 0, 30, 50 mg/kg/d); 2-methoxyethanol (2-ME; 0, 75, 150 mg/kg/d), or valproic acid (VPA; 0, 125, 250 mg/kg/d). Incidences of SNR were 9.3-27.6% in controls and 19.3-84.4% in the high dosage groups. SNR length showed a bimodal distribution with peaks at 0.3-0.4 mm and 0.9-1.1 mm in both treated and control groups. Based on length distributions, we used an actual length of 0.6 mm to separate short (rudimentary) from long (extra) SNR. DIN, 2-ME, and VPA induced a dose-related increase of extra ribs, while the incidence of rudimentary ribs remained at control levels. There was no apparent correlation of the presence of either type of SNR in a fetus and the occurrence of other anomalies. These data support the idea that extra and rudimentary SNR may reflect separate developmental phenomena, and should be considered and reported separately in developmental toxicity studies for risk assessment.     

p21-Activated kinase 5 (Pak5) localizes to mitochondria and inhibits apoptosis by phosphorylating BAD

Pak5 is the most recently identified and least understood member of the p21-activated kinase (Pak) family. This kinase is known to promote neurite outgrowth in vitro, but its localization, substrates, and effects on cell survival have not been reported. We show here that Pak5 has unique properties that distinguish it from all other members of the Pak family. First, Pak5, unlike Pak1, cannot complement an STE20 mutation in Saccharomyces cerevisiae. Second, Pak5 binds to the GTPases Cdc42 and Rac, but these GTPases do not regulate Pak5 kinase activity, which is constitutive and stronger than any other Pak. Third, Pak5 prevents apoptosis induced by camptothecin and C2-ceramide by phosphorylating BAD on Ser-112 in a protein kinase A-independent manner and prevents the localization of BAD to mitochondria, thereby inhibiting the apoptotic cascade that leads to apoptosis. Finally, we show that Pak5 itself is constitutively localized to mitochondria, and that this localization is independent of kinase activity or Cdc42 binding. These features make Pak5 unique among the Pak family and suggest that it plays an important role in apoptosis through BAD phosphorylation.     

Lentivirus Nef specifically activates Pak2

Nef proteins from human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) have been found to associate with an active cellular serine/threonine kinase designated Nef-associated kinase (Nak). The exact identity of Nak remains controversial, with two recent studies indicating that Nak may be either Pak1 or Pak2. In this study, we investigated the hypothesis that such discrepancies arise from the use of different Nef alleles or different cell types by individual investigators. We first confirm that Pak2 but not Pak1 is cleaved by caspase 3 in vitro and then demonstrate that Nak is caspase 3 sensitive, regardless of Nef allele or cell type used. We tested nef alleles from three lentiviruses (HIV-1 SF2, HIV-1 NL4-3, and SIVmac239) and used multiple cell lines of myeloid, lymphoid, and nonhematopoietic origin to evaluate the identity of Nak. We demonstrate that ectopically expressed Pak2 can substitute for Nak, while ectopically expressed Pak1 cannot. We then show that Nef specifically mediates the robust activation of ectopically expressed Pak2, directly demonstrating that Nef regulates Pak2 activity and does not merely associate with activated Pak2. We report that most of the active Pak2 is found bound to Nef, although a fraction is not. In contrast, only a small amount of Nef is found associated with Pak2. We conclude that Nak is Pak2 and that Nef specifically mediates Pak2 activation in a low-abundance complex. These results will facilitate both the elucidation of the role of Nef in pathogenesis and the development of specific inhibitors of this highly conserved function of Nef.     

Regulation of macropinocytosis by p21-activated kinase-1

The process of macropinocytosis is an essential aspect of normal cell function, contributing to both growth and motile processes of cells. p21-activated kinases (PAKs) are targets for activated Rac and Cdc42 guanosine 5'-triphosphatases and have been shown to regulate the actin-myosin cytoskeleton. In fibroblasts PAK1 localizes to areas of membrane ruffling, as well as to amiloride-sensitive pinocytic vesicles. Expression of a PAK1 kinase autoinhibitory domain blocked both platelet-derived growth factor- and RacQ61L-stimulated uptake of 70-kDa dextran particles, whereas an inactive version of this domain did not, indicating that PAK kinase activity is required for normal growth factor-induced macropinocytosis. The mechanisms by which PAK modulate macropinocytosis were examined in NIH3T3 cell lines expressing various PAK1 constructs under the control of a tetracycline-responsive transactivator. Cells expressing PAK1 (H83,86L), a mutant that dramatically stimulates formation of dorsal membrane ruffles, exhibited increased macropinocytic uptake of 70-kDa dextran particles in the absence of additional stimulation. This effect was not antagonized by coexpression of dominant-negative Rac1-T17N. In the presence of platelet-derived growth factor, both PAK1 (H83,86L) and a highly kinase active PAK1 (T423E) mutant dramatically enhanced the uptake of 70-kDa dextran. Neither wild-type PAK1 nor vector controls exhibited enhanced macropinocytosis, nor did PAK1 (H83,86L) affect clathrin-dependent endocytic mechanisms. Active versions of PAK1 enhanced both growth factor-stimulated 70-kDa dextran uptake and efflux, suggesting that PAK1 activity modulated pinocytic vesicle cycling. These data indicate that PAK1 plays an important regulatory role in the process of macropinocytosis, perhaps related to the requirement for PAK in directed cell motility.     

Down-regulation of insulin signaling by protein-tyrosine phosphatase 1B is mediated by an N-terminal binding region

Protein-tyrosine phosphatases (PTPs) play a major role in regulating insulin signaling. Among the PTPs that regulate this signaling pathway, PTP1B plays an especially prominent role. PTP1B inhibits insulin signaling and has previously been shown to bind to the activated insulin receptor (IR), but neither the mechanism nor the physiological importance of such binding have been established. Here, we show that a previously undefined region in the N-terminal, catalytic half of PTP1B contributes to IR binding. Point mutations within this region of PTP1B disrupt IR binding but do not affect the catalytic activity of this phosphatase. This binding-defective mutant of PTP1B does not efficiently dephosphorylate the IR in cells, nor does it effectively inhibit IR signaling. These results suggest that PTP1B targets the IR through a novel binding element and that binding is required for the physiological effects of PTP1B on IR signal transduction.     

Evolutionary conservation of prion-forming abilities of the yeast Sup35 protein

Saccharomyces cerevisiae prion [PSI ] is a self-propagating isoform of the eukaryotic release factor eRF3 (Sup35p). Sup35p consists of the evolutionary conserved release factor domain (Sup35C) and two evolutionary variable regions - Sup35N, which serves as a prion-forming domain in S. cerevisiae, and Sup35M. Here, we demonstrate that the prion form of Sup35p is not observed among industrial and natural strains of yeast. Moreover, the prion ([PSI + ]) state of the endogenous S. cerevisiae Sup35p cannot be transmitted to the next generations via heterologous Sup35p or Sup35NM, originating from the distantly related yeast species Pichia methanolica. This suggests the existence of a 'species barrier' in yeast prion conversion. However, the chimeric Sup35p, containing the Sup35NM region of Pichia, can be turned into a prion in S. cerevisiae by overproduction of the identical Pichia Sup35NM. Therefore, the prion-forming potential of Sup35NM is conserved in evolution. In the heterologous system, overproduction of Pichia Sup35p or Sup35NM induced formation of the prion form of S. cerevisiae Sup35p, albeit less efficiently than overproduction of the endogenous Sup35p. This implies that prion induction by protein overproduction does not require strict correspondence of the 'inducer' and 'inducee' sequences, and can overcome the 'species barrier'.