A Ca2+-Dependent Protein Kinase Activity Associated with Serotonin Binding Protein

The endogenous phosphorylation of serotonin binding protein (SBP), a soluble protein found in central and peripheral serotonergic neurons, inhibits the binding of 5-hydroxytryptamine (5-HT, serotonin). A protein kinase activity that copurifies with SBP (SBP-kinase) was partially characterized and compared with calcium/calmodulin-dependent protein kinase II (CAM-PK II). SBP itself is not the enzyme since heating destroyed the protein kinase activity without affecting the capacity of the protein to bind [3H]5-HT. SBP-kinase and CAM-PK II kinase shared the following characteristics: (1) size of the subunits; (2) autophosphorylation in a Ca2+-dependent manner; and (3) affinity for Ca2+. In addition, both forms of protein kinase phosphorylated microtubule-associated proteins well and did not phosphorylate myosin, phosphorylase b, and casein. Phorbol esters or diacylglycerol had no effect on either of the protein kinases. However, substantial differences between SBP-kinase and CAM-PK II were observed: (1) CAM enhanced CAM-PK II activity, but had no effect on SBP-kinase; (2) synapsin I was an excellent substrate for CAM-PK II, but not for SBP-kinase; (3) 5-HT inhibited both the autophosphorylation of SBP-kinase and the phosphorylation of SBP, but had no effect on CAM-PK II. These data indicate that SBP-kinase is different from CAM-PK II. Phosphopeptide maps of SBP and SBP-kinase generated by digestion with S. aureus V8 protease are consistent with the conclusion that these proteins are distinct molecular entities. It is suggested that phosphorylation of SBP may regulate the transport of 5-HT within neurons.     

Factors involved in multiplication and survival ofEscherichia coli in lake water

The population of a strain ofEscherichia coli that was resistant to nalidixic acid and streptomycin declined rapidly in samples of sterile and nonsterile Cayuga Lake water and reached an undetectable level in nonsterile water at 24 and 72 hours when counted on eosin-methylene blue (EMB) agar and half-strength trypticase soy agar (TSA), respectively. In sterile lake water amended with 10g amino acids per ml or 0.1 M phosphate,E. coli multiplied exponentially for more than 24 hours. The addition ofRhizobium leguminosarum biovarphaseoli to unamended sterile lake water prevented the decline ofE. coli, and its addition to amended sterile lake water preventedE. coli multiplication. The cell density of this strain ofE. coli declined in the first 8 hours after its introduction into an inorganic salts solution, but the bacterium then grew extensively. This increase in abundance was not observed in the presence ofR. phaseoli, andE. coli counts on half-strength TSA remained unchanged between 8 hours and 6 days. When counted on EMB agar, the abundance of the antibiotic-resistant strain ofE. coli and a strain not selected for resistance increased in solutions containing phosphate and amino acids but declined in the presence of high densities ofR. phaseoli. Many of the cells of the antibiotic-resistantE. coli strain failed to grow on antibiotic-amended EMB agar after introduction of the organism into nonsterile or sterile lake water or into an inorganic salts solution containingR. phaseoli, although colonies appeared on TSA. The data suggest thatE. coli cells grown on rich media suffer a shock when introduced into lake water because of low hypotonicity, the indigenous competing flora, or both. This shock is prevented by either phosphate buffer or by amino acids at low concentration. The shocked bacteria formed colonies on half-strength TSA. Depending on environmental conditions, the presence of a second organism either has no effect or results in an increase or decrease inE. coli numbers.     

The structure of the incompatibility factors of Schizophyllum commune: Constitution of the three classes of B factors

Summary The B factors of Schizophyllum commune are of 3 classes: The high recombining class I has 7 alleles and 7 alleles; the low recombining classes are class II with 7 allels and probably 2 alleles and class III with probably 2 (or also 2) alleles and 7 allels. A fourth hypothetical class (-) was not found and either does not exist or is indistinguishable from class III by the tests employed. The and alleles differ from and by either (a) mutations affecting both mating specificity and recombination frequency, or (b) deletions involving most of the B region.The research was supported by a grant from the Atomic Energy Commission of the U.S. No. (30-1)-3875 and was performed at the Biological Laboratories, Harvard University, Cambridge, Mass., U.S.A.     

The genetic structure of the incompatibility factors of Schizophyllum commune: the resolution of class 3 B factors

Summary Recombinant B factors were recovered from crosses of class Ixclass III B factors. Factors of class III are constituted of two loci, but intrafactor recombination occurs at a low frequency. B of class III B factors differs in its properties from B of class I, but B is identical with B of class I. The B locus has at least two alleles.     

Survival of six species of African ticks in relation to saturation deficits

The survival of unfed males and females of six species of African ticks was monitored at five different saturation deficits at constant temperature (25°C). The survivorship curves for each species comprised a pre-mortality period, prior to when ticks started to die and a mortality period corresponding to a rapid increase in the mortality rate. Longevity was defined as pre-mortality plus mortality. A negative correlation between the longevity of the ticks and the saturation deficits was found with ticks surviving longer at lower deficits. The survival of males and females was similar. At low saturation deficits (2–4 mmHg) Amblyomma hebraeum survived the longest periods (74 weeks). Some correlation was found between the tick survival under dehydrating conditions and habitat associations. Rhipicephalus appendiculatus and Haemaphysalis leachii, the most mesic in distribution, had the shortest longevity (21 and 13 weeks, respectively) at high saturation deficits (7–21 mmHg). Hyalomma marginatum rufipes, the most xerophilic in distribution, had the longest survival (39.3±10.5 weeks) at high saturation deficits. Other factors apart from the adult survival should be taken into account when accounting for the tick distribution, in particular the tolerance of earlier developmental stages to desiccation.     

Homologs of the yeast neck filament associated genes: isolation and sequence analysis of Candida albicans CDC3 and CDC10

Morphogenesis in the yeast Saccharomyes cerevisiae consists primarily of bud formation. Certain cell division cycle (CDC) genes, CDC3, CDC10, CDC11, CDC12, are known to be involved in events critical to the pattern of bud growth and the completion of cytokinesis. Their products are associated with the formation of a ring of neck filaments that forms at the region of the mother cell-bud junction during mitosis. Morphogenesis in Candida albicans, a major fungal pathogen of humans, consists of both budding and the formation of hyphae. The latter is thought to be related to the pathogenesis and invasiveness of C. albicans. We have isolated and characterized C. albicans homologs of the S. cerevisiae CDC3 and CDC10 genes. Both C. albicans genes are capable of complementing defects in the respective S. cerevisiae genes. RNA analysis of one of the genes suggests that it is a regulated gene, with higher overall expression levels during the hyphal phase than in the yeast phase. Not surprisingly, DNA sequence analysis reveals that the proteins share extensive homology at the amino acid level with their respective S. cerevisiae counterparts. Related genes are also found in other species of Candida and, more importantly, in filamentous fungi such as Aspergillus nidulans and Neurospora crassa. A database search revealed significant sequence similarity with two peptides, one from Drosophila and one from mouse, suggesting strong evolutionary conservation of function.     

Ecto-Protein Kinase and Surface Protein Phosphorylation in PC12 Cells: Interactions with Nerve Growth Factor

Abstract: The phosphorylation of surface proteins by ectoprotein kinase has been proposed to play a role in mechanisms underlying neuronal differentiation and their responsiveness to nerve growth factor (NGF). PC 12 clones represent an optimal model for investigating the mode of action of NGF in a homogeneous cell population. In the present study we obtained evidence that PC12 cells possess ectoprotein kinase and characterized the endogenous phosphorylation of its surface protein substrates. PC12 cells maintained in a chemically defined medium exhibited phosphorylation of proteins by [γ-³²P]ATP added to the medium at time points preceding the intracellular phosphorylation of proteins in cells labeled with ³²P_i. This activity was abolished by adding apyrase or trypsin to the medium but was not sensitive to addition of an excess of unlabeled P_i. As also expected from ecto-protein kinase activity, PC12 cells catalyzed the phosphorylation of an exogenous protein substrate added to the medium, dephospho-α-casein, and this activity competed with the endogenous phosphorylation for extracellular ATP. Based on these criteria, three protein components migrating in sodium dodecyl sulfate gels with apparent molecular weights of 105K, 39K, and 20K were identified as exclusive substrates of ecto-protein kinase in PC12 cells. Of the phosphate incorporated into these proteins from extracellular ATP, 75–87% was found in phosphothreonine. The phosphorylation of the 39K protein by ecto-protein kinase did not require Mg²⁺, implicating this activity in the previously demonstrated regulation of Ca²⁺-dependent, high-affinity norepinephrine uptake in PC12 cells by extracellular ATP. The protein kinase inhibitor K-252a inhibited both intra- and extracellular protein phosphorylation in intact PC12 cells. Its hydrophilic analogue K-252b, had only minimal effects on intracellular protein phosphorylation but readily inhibited the phosphorylation of specific substrates of ecto-protein kinase in PC12 cells incubated with extracellular ATP, suggesting the involvement of ecto-protein kinase in the reported inhibition of NGF-induced neurite extension by K-252b. Preincubation of PC12 cells with 50 ng/ml of NGF for 5 min stimulated the activity of ecto-protein kinase toward all its endogenous substrates. Exposure of PC12 cells to the same NGF concentration for 3 days revealed another substrate of ecto-protein kinase, a 53K protein, whose surface phosphorylation is expressed only after NGF-induced neuronal differentiation. In the concentration range (10–100 μM) at which 6-thioguanine blocked NGF-promoted neurite outgrowth in PC12 cells, 6-thioguanine effectively inhibited the phosphorylation of specific proteins by ecto-protein kinase. This study provides the basis for continued investigation of the involvement of ecto-protein kinase and its surface protein substrates in neuronal differentiation, neuritogenesis, and synaptogenesis.     

Dominant missense mutations in a novel yeast protein related to mammalian phosphatidylinositol 3-kinase and VPS34 abrogate rapamycin cytotoxicity.

Rapamycin is a macrolide antifungal agent that exhibits potent immunosuppressive properties. In Saccharomyces cerevisiae, rapamycin sensitivity is mediated by a specific cytoplasmic receptor which is a homolog of human FKBP12 (hFKBP12). Deletion of the gene for yeast FKBP12 (RBP1) results in recessive drug resistance, and expression of hFKBP12 restores rapamycin sensitivity. These data support the idea that FKBP12 and rapamycin form a toxic complex that corrupts the function of other cellular proteins. To identify such proteins, we isolated dominant rapamycin-resistant mutants both in wild-type haploid and diploid cells and in haploid rbp1::URA3 cells engineered to express hFKBP12. Genetic analysis indicated that the dominant mutations are nonallelic to mutations in RBP1 and define two genes, designated DRR1 and DRR2 (for dominant rapamycin resistance). Mutant copies of DRR1 and DRR2 were cloned from genomic YCp50 libraries by their ability to confer drug resistance in wild-type cells. DNA sequence analysis of a mutant drr1 allele revealed a long open reading frame predicting a novel 2470-amino-acid protein with several motifs suggesting an involvement in intracellular signal transduction, including a leucine zipper near the N terminus, two putative DNA-binding sequences, and a domain that exhibits significant sequence similarity to the 110-kDa catalytic subunit of both yeast (VPS34) and bovine phosphatidylinositol 3-kinases. Genomic disruption of DRR1 in a mutant haploid strain restored drug sensitivity and demonstrated that the gene encodes a nonessential function. DNA sequence comparison of seven independent drr1dom alleles identified single base pair substitutions in the same codon within the phosphatidylinositol 3-kinase domain, resulting in a change of Ser-1972 to Arg or Asn. We conclude either that DRR1 (alone or in combination with DRR2) acts as a target of FKBP12-rapamycin complexes or that a missense mutation in DRR1 allows it to compensate for the function of the normal drug target.