Zeke T et al. (1996) Worm Breeder's Gazette "Detection of Ser/Thr protein phosphatase activities in C.elegans"

[

Worm Breeder's Gazette,

1996]

Protein phosphatases (PP) are significant regulatory enzymes of all eukaryotes. They must be present in C.elegans as witnessed by DNA-deduced PP-like amino acid sequences unraveledin the framework of the genom project. About half of them belong to Ser/Thr PP, while the rest is Tyr PP. For the assay of the main Ser/Thr PP classes, namely PP1, PP2A, PP2B and PP2C Cohen and coworkers suggested a straightforward and relatively simple procedure (1) that was adopted for the analysis of the worm. Rabbit muscle phosphorylase a phosphorylated by phospho- rylase kinase, rabbit muscle inhibitor-1 and casein from bovine milk phosphorylated by bovine heart cAMP-dependent protein kinase were used as substrates. C.elegans Var. Bristol (N2) wild type strain were cultured, harvested, rinsed with the extraction buffer, frozen in liquid nitrogen and homogenized in four volumes of the ice cold extraction buffer (50 mM Tris-HCl pH=7.4; 0.1 mM EDTA; 10 mM DTT; 0.5% Triton X-100; 2mM PMSF; 5mM benzamidine and 1 mM o-phenantroline).The homogenates were cleared by cent- rifugation at 14000 g, 4 oC for 10 min and than were kept frozen in liquid nitrogen till the assays. Rabbit skeletal muscle inhibitor-2 (unphosphorylated) was used as a specific inhibitor ofPP1 with 32P-phosphorylase a substrate in the presence ofEDTA,a metal ion chelator(Fig.1).The titration curve reveals that about 75%of the total activity was supplied by PP1 and 5 nM of inhibitor-2 caused about 50% inhibition. Okadaic acid, a potent marine toxin and tumor promoter, was used to separate PP1 and PP2A activities with 32P-phosphorylase a substrate in the pre- sence of EDTA (Fig.2). The titration curve with okadaic acid is bi- phasic, in the first step about 30% of the total activity is inhibited with an IC50 of ~0.04 nM and the rest of activity was inhibited in a second step with an IC50 of ~70 nM. Complete inhibition was achieved by 1 micromol/liter okadaic acid concentration. It is known that PP2A is more sensitive to okadaic acid than PP1, thus the first step of the titration can be explained by the presence of PP2A while the second step can be attributed to PP1 inhibition. For the detection of PP2B we used 32P-inhibitor-1 substrate and Ca-calmodulin as an activator. Due to the interference of prote- ases the 32Pi liberated in the assays was estimated after ammonium molybdate extraction(2). About 50 nmol substrate was converted in 1 min by the extract. ~70% of the activity was inhibited by 4 mM EDTA or 1.5 mM trifluoperazine and ~30% was inhibited by 2 nM okadaic acid (not shown). Thus 70% of the total activity was due to PP2B and 30% to PP2A. PP2C was assayed with 32P-casein substrate in the presence of Mg2+-activator. ~10%of the activity was inhibited by inhibitor-2 (PP1) ~40% of the activity was inhibited by 2 nM okadaic acid (PP2A) and ~50% was stimulated by Mg2+ (PP2C). Half maximal activation was achieved at 2 mM Mg2+ concentration, maximal activation was measured at 20 mM (Fig.3.). Acknowledgements.This work was supported by the grants OTKA 6005 and 12840. (1) Cohen, P., Klump, S., Schelling, D.L. (1989) FEBS Lett. 250, 596-600. (2) Shenolikar, S., Ingebritsen, T.S. (1984) Meth. Enyzmol. 107, 103-129.

Mirani G et al. (1998) Worm Breeder's Gazette "Sma-9 a Novel Gene in TGFb Signaling; Something Strange about him-8 Males"

Hao Y, Mirani G, Savage C

[

Worm Breeder's Gazette,

1998]

We are interested in understanding TGFb signaling pathway. Work in several laboratories has led to a model in which Smad proteins are activated by phosphorylation by TGFb receptors allowing them to translocate to the nucleus and act as transcriptional activators. There is a continued interest in identifying new components of this pathway. Sma-9 is a putative member of this pathway. Complementation tests have shown that there are four alleles of sma-9 — wk55, wk62, wk71, and wk82. Another sma-9 allele, wk121, was kindly given to us by S. Cohen and R. Padgett. Genetic screen described below has yielded us with one more allele of sma-9, which is named qc1. Sma-9 expresses the classical phenotype expressed by other sma genes. It affects body size and male tail ray patterning. Sma-9 mutant animals are small in size. We have examined males for the four sma-9 alleles (wk55, wk62, wk71, and wk82) for ray patterning defects (see Table 1 for that). To increase the frequency of males, we made him-5(e1490);sma-9 doubles for all four of sma-9 alleles. In fact, we had initially made doubles with him-8(e1467). However, Scott Emmons had pointed out to us at the Worm Meeting that this him-8 strain has males that are not wild type. They have ray patterning defects of their own. We checked by examining about seven male tail sides using the Nomaraski Microscopy. They all had fusions of rays 8 and 9. And strangely enough, our sma-9 males also exhibit fusions of rays 8 and 9. We also examined him-5 males. Even they exhibited fusions of rays 8 and 9, however, at a lower frequency. However, this fusion of 8 and 9 is different from the one exhibited by sma-9 males. In him-5 and him-8 males, when rays 8 and 9 are fused, the fused ray resembles ray 9, whereas in the sma-9 fusion of rays 8 and 9, the fused ray resembles ray 8. Besides male tail characterization, we are also interested in generating new mutations in sma-9. So far, we have done six EMS mutagenesis experiments. Mutagenized N2 males were crossed with sma-9unc-7 hermaphrodites. And in the F1 generation, we scored non Unc hermaphrodites. We have obtained one new sma-9 allele, qc1 from 5700 chromosomes scored. The complementation test has shown that it is in fact, a new sma-9 allele.