The BAM1/BAM2 receptor-like kinases are important regulators of Arabidopsis early anther development

Anther development involves the formation of several adjacent cell types required for normal male fertility. Only a few genes are known to be involved in early anther development, particularly in the establishment of these different cell layers. Arabidopsis thaliana BAM1 (for BARELY ANY MERISTEM) and BAM2 encode CLAVATA1-related Leu-rich repeat receptor-like kinases that appear to have redundant or overlapping functions. We characterized anther development in the bam1 bam2 flowers and found that bam1 bam2 anthers appear to be abnormal at a very early stage and lack the endothecium, middle, and tapetum layers. Analyses using molecular markers and cytological techniques of bam1 bam2 anthers revealed that cells interior to the epidermis acquire some characteristics of pollen mother cells (PMCs), suggesting defects in cell fate specification. The pollen mother-like cells degenerate before the completion of meiosis, suggesting that these cells are defective. In addition, the BAM1 and BAM2 expression pattern supports both an early role in promoting somatic cell fates and a subsequent function in the PMCs. Therefore, analysis of BAM1 and BAM2 revealed a cell-cell communication process important for early anther development, including aspects of cell division and differentiation. This finding may have implications for the evolution of multiple signaling pathways in specifying cell types for microsporogenesis.     

Anisomycin-activated protein kinases p45 and p55 but not mitogen-activated protein kinases ERK-1 and -2 are implicated in the induction of c-fos and c-jun.

Independent of its ability to block translation, anisomycin intrinsically initiates intracellular signals and immediate-early gene induction [L. C. Mahadevan and D. R. Edwards, Nature (London) 349:747-749, 1991]. Here, we characterize further its action as a potent, selective signalling agonist. In-gel kinase assays show that epidermal growth factor (EGF) transiently activates five kinases: the mitogen-activated protein (MAP) kinases ERK-1 and -2, and three others, p45, p55, and p80. Anisomycin, at inhibitory and subinhibitory concentrations, does not activate ERK-1 and -2 but elicits strong sustained activation of p45 and p55, which are unique in being serine kinases whose detection is enhanced with poly-Glu/Tyr or poly-Glu/Phe copolymerized in these gels. Translational arrest using emetine or puromycin does not activate p45 and p55 but does prolong EGF-stimulated ERK-1 and -2 activation. Rapamycin, which blocks anisomycin-stimulated p70/85S6k activation without affecting nuclear responses, has no effect on p45 or p55 kinase. p45 and p55 are activable by okadaic acid or UV irradiation, and both kinases phosphorylate the c-Jun NH2-terminal peptide 1-79, putatively placing them within c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) subfamily of MAP kinases. Thus, the EGF- and anisomycin-activated kinases p45 and p55 are strongly implicated in signalling to c-fos and c-jun, whereas the MAP kinases ERK-1 and -2 are not essential for this process.     

Fcf1p and Fcf2p are novel nucleolar Saccharomyces cerevisiae proteins involved in pre-rRNA processing

The uncharacterized Saccharomyces cerevisiae proteins Fcf1 and Fcf2, encoded by the ORFs YDR339c and YLR051c, respectively, were identified in a tandem affinity purification experiment of the known 40S factor Faf1p. Most of the proteins associated with TAP-Faf1p are trans-acting factors involved in pre-rRNA processing and 40S subunit biogenesis, in agreement with the previously observed role of Faf1p in 18S rRNA synthesis. Fcf1p and Fcf2p are both essential and localize to the nucleolus. Depletion of Fcf1p and Fcf2p leads to a decrease in synthesis of the 18S rRNA, resulting in a deficit in 40S ribosomal subunits. Northern analysis indicates inefficient processing of pre-rRNA at the A(0), A(1), and A(2) cleavage sites.     

CLO/GFA1 and ATO are novel regulators of gametic cell fate in plants

The formation of gametes is a key step in the life cycle of any sexually reproducing organism. In flowering plants, gametes develop in haploid structures termed gametophytes that comprise a few cells. The female gametophyte forms gametic cells and flanking accessory cells. During a screen for regulators of egg-cell fate, we isolated three mutants, lachesis (lis), clotho (clo) and atropos (ato), that show deregulated expression of an egg-cell marker. We have previously shown that, in lis mutants, which are defective for the splicing factor PRP4, accessory cells can differentiate gametic cell fate. Here, we show that CLOTHO/GAMETOPHYTIC FACTOR 1 (CLO/GFA1) is necessary for the restricted expression of egg- and central-cell fate and hence reproductive success. Surprisingly, infertile gametophytes can be expelled from the maternal ovule tissue, thereby preventing the needless allocation of maternal resources to sterile tissue. CLO/GFA1 encodes the Arabidopsis homologue of Snu114, a protein that is considered to be an essential component of the spliceosome. In agreement with their proposed role in pre-mRNA splicing, CLO/GFA1 and LIS co-localize to nuclear speckles. Our data also suggest that CLO/GFA1 is necessary for the tissue-specific expression of LIS. Furthermore, we demonstrate that ATO encodes the Arabidopsis homologue of SF3a60, a protein that has been implicated in pre-spliceosome formation. Our results thus establish that the restriction of gametic cell fate is specifically coupled to the function of various core spliceosomal components.     

Membrane-associated cytoskeleton and transbilayer phospholipid asymmetry

Earlier studies have suggested that the membrane-associated cytoskeleton (membrane skeleton) in erythrocytes plays a major role in maintaining the transmembrane phospholipid asymmetry. But recently, it has been proposed that an ATP-dependent aminophospholipid pump is the sole determinant of this asymmetry in these cells. A critical analysis of the published data along with some unpublished results from the author's laboratory, however, indicate that both membrane skeleton and ATP-dependent aminophospholipid pump are required for maintaining the membrane phospholipid asymmetry in native erythrocytes.     

Cyclin D2 and p27 are tissue-specific regulators of tumorigenesis in inhibin alpha knockout mice

Inhibins are heterodimeric (alpha:betaA and alpha:betaB) endocrine, paracrine, and autocrine factors of the TGFbeta superfamily that are produced predominantly by ovarian granulosa cells in females and testicular Sertoli cells in males. Control of granulosa and Sertoli cell proliferation is lost in the inhibin alpha (Inhalpha) knockout mouse model, leading to gonadotropin-dependent gonadal tumors of the granulosa/Sertoli cell lineage in both females and males. Castrate Inhalpha knockout mice develop sex steroidogenic tumors of the adrenal cortex. Physiological control of granulosa/Sertoli cell cycle progression depends on p27Kip1 and cyclin D2, which function in the G1-->S phase transition. To study the cell cycle-regulatory factors involved in ovarian, testicular, and adrenal tumor development in vivo, we have bred Inhalpha mutant mice to mice with targeted disruptions of the p27 and cyclin D2 genes. Our previous studies demonstrated that inhibins act cooperatively with p27 to negatively regulate granulosa cell proliferation, as double mutant mice lacking inhibins and p27 develop and succumb to ovarian tumors more rapidly than Inhalpha knockout mice. Here, we report that cyclin D2 antagonizes this inhibition and is key in promoting gonadal growth and tumor development, and tumor development is markedly suppressed in double-mutant mice. We found that double-knockout females lacking cyclin D2 and Inhalpha lived longer than mice lacking inhibins alone; the majority of these double-knockout mice lived longer than 17 wk, as opposed to inhibin alpha single-knockout females with 50% survival at between 12 and 13 wk of age. Moreover, 95% of inhibin alpha knockout males succumb to testicular tumor development by 12 wk of age, whereas double knockouts were protected from early signs of tumor development and had a 50% survival of 40 wk. Interestingly, the results of these studies reflect tissue-specific consequences of loss of these cell cycle regulators. In castrate mice, loss of p27 has little effect on adrenal cortical tumor progression in the absence of inhibins, whereas loss of cyclin D2 prolongs the lifespan of cyclin D2, Inhalpha double knockouts. After gonadectomy, 50% of cyclin D2, Inhalpha double-knockout males live to more than 46 wk of age, 10 wk longer than 50% of littermates lacking only inhibins. Similarly, 50% of female cyclin D2, inhibin alpha double knockouts live to 47 wk of age before succumbing to adrenal tumor development, in contrast to the 50% survival of Inhalpha single-knockout females at between 27 and 28 wk. Thus, identification of genetic modifiers of the Inhalpha knockout tumor phenotype has led us to a better appreciation of how specific components of the cell cycle machinery contribute to tumorigenesis in the ovary, testis, and adrenal gland.     

Cdk-inhibitory activity and stability of p27Kip1 are directly regulated by oncogenic tyrosine kinases

p27Kip1 controls cell proliferation by binding to and regulating the activity of cyclin-dependent kinases (Cdks). Here we show that Cdk inhibition and p27 stability are regulated through direct phosphorylation by tyrosine kinases. A conserved tyrosine residue (Y88) in the Cdk-binding domain of p27 can be phosphorylated by the Src-family kinase Lyn and the oncogene product BCR-ABL. Y88 phosphorylation does not prevent p27 binding to cyclin A/Cdk2. Instead, it causes phosphorylated Y88 and the entire inhibitory 3(10)-helix of p27 to be ejected from the Cdk2 active site, thus restoring partial Cdk activity. Importantly, this allows Y88-phosphorylated p27 to be efficiently phosphorylated on threonine 187 by Cdk2 which in turn promotes its SCF-Skp2-dependent degradation. This direct link between transforming tyrosine kinases and p27 may provide an explanation for Cdk kinase activities observed in p27 complexes and for premature p27 elimination in cells that have been transformed by activated tyrosine kinases.     

Multidomain STS/TULA proteins are novel cellular regulators

Proteins of the STS/TULA family recently emerged as important regulators of cellular functions. They exhibit a unique domain architecture, featuring at least three interactive/functional domains. Despite a significant degree of homology between the two members of this family, there are considerable functional differences between them. Thus, one of them is ubiquitously expressed in mammalian tissues and exhibits high phosphatase activity, whereas the other one is expressed in lymphocytes only and exhibits very low phosphatase activity, but is capable of promoting apoptosis, an activity unique for this family member. Among several functions reported for STS/TULA proteins, the most characterized one is the regulation of protein tyrosine kinase-mediated signaling. Interestingly, gene deletion of neither family member results in a discernible phenotype, whereas simultaneous deletion of both members causes hyperreactivity of T cells. Despite their apparent importance, the physiological role and the molecular basis of the effects of STS/TULA proteins remain poorly understood. This brief review summarizes what is currently known about the STS/TULA family and outlines the unresolved questions in this area.     

The Cdc42p GTPase and its regulators Nrf1p and Scd1p are involved in endocytic trafficking in the fission yeast Schizosaccharomyces pombe

Nrf1p was first identified in a screen for negative regulators of the Cdc42p GTPase. Overexpression of Nrf1p resulted in dose-dependent lethality, with cells exhibiting an ellipsoidal morphology and abnormal vacuolar phenotypes including an increase in vacuolar fusion. Green fluorescent protein (GFP)-Cdc42p and GFP-Nrf1p colocalized to vacuolar membranes and GFP-Nrf1p vacuolar localization depended on Scd1p, the Schizosaccharomyces pombe homolog of the Cdc24p guanine nucleotide exchange factor. In this study, site-directed mutagenesis was conducted on Nrf1p to determine its functional domains. Mutations in the three putative transmembrane domains resulted in mislocalization of GFP-Nrf1p and an inability to induce lethality, suggesting a loss of function. Mutations in the second extramembranous loop of Nrf1p also resulted in a loss of function and altered the ability of GFP-Nrf1p to localize to vacuolar membranes. Analysis of Deltanrf1 and Deltascd1 mutants revealed defects in endocytosis. In addition, overexpression of constitutively active Cdc42(G12V)p resulted in an increase in endocytosis and an ability to rescue the endocytic defects in Deltanrf1 and Deltascd1 cells. These data are consistent with Nrf1p and Scd1p being necessary for efficient endocytosis, possibly through the regulation of Cdc42p.     

Drs2p-related P-type ATPases Dnf1p and Dnf2p are required for phospholipid translocation across the yeast plasma membrane and serve a role in endocytosis

Plasma membranes in eukaryotic cells display asymmetric lipid distributions with aminophospholipids concentrated in the inner and sphingolipids in the outer leaflet. This asymmetry is maintained by ATP-driven lipid transporters whose identities are unknown. The yeast plasma membrane contains two P-type ATPases, Dnf1p and Dnf2p, with structural similarity to ATPase II, a candidate aminophospholipid translocase from bovine chromaffin granules. Loss of Dnf1p and Dnf2p virtually abolished ATP-dependent transport of NBD-labeled phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine from the outer to the inner plasma membrane leaflet, leaving transport of sphingolipid analogs unaffected. Labeling with trinitrobenzene sulfonic acid revealed that the amount of phosphatidylethanolamine exposed on the surface of Deltadnf1Deltadnf2 cells increased twofold relative to wild-type cells. Phosphatidylethanolamine exposure by Deltadnf1Deltadnf2 cells further increased upon removal of Drs2p, an ATPase II homolog in the yeast Golgi. These changes in lipid topology were accompanied by a cold-sensitive defect in the uptake of markers for bulk-phase and receptor-mediated endocytosis. Our findings demonstrate a requirement for Dnf1p and Dnf2p in lipid translocation across the yeast plasma membrane. Moreover, it appears that Dnf1p, Dnf2p and Drs2p each help regulate the transbilayer lipid arrangement in the plasma membrane, and that this regulation is critical for budding endocytic vesicles.     

Histidine kinases and response regulators in networks

Two-component systems, composed of a histidine kinase (HK) and a response regulator (RR), are the major signal transduction devices in bacteria. Originally it was thought that these two components function as linear, phosphorylation-driven stimulus-response system. Here, we will review how accessory proteins are employed by HKs and RRs to mediate signal integration, scaffolding, interconnection and allosteric regulation, and how these two components are embedded in regulatory networks.