Protein-tyrosine phosphatase activity of hairy cell tartrate-resistant acid phosphatase.

Tartrate-resistant acid phosphatase (TRAcP) is a reliable cytochemical marker for the diagnosis of hairy cell leukemia (HCL). The enzyme has been the subject of much biochemical investigation yet its function in the hairy cells (HC) is still unknown. Two TRAcPs have been purified from HCL spleen tissues by a series of chromatographic separations. The two enzymes, provisionally called peak 1 and peak 2, had specific activities of greater than 600 U/mg and 800 U/mg respectively when p-nitrophenyl phosphate (p-NPP) was used as substrate and had Km values in the range of 1 to 5 mM p-NPP. The two TRAcPs had the same substrate specificities and inhibitor sensitivities, therefore could be isoforms of the same enzyme. Their pH optima were between 5 and 6 for all substrates tested including the phosphotyrosine-containing peptide, Raytide, which was still hydrolyzed efficiently at neutral pH. Neither phosphoserine nor phosphoserine-containing casein were hydrolyzed by either enzyme. The TRAcPs of HC may thus be capable of functioning as protein-tyrosine phosphatases (PTP). High activity of a PTP could regulate the activities of protein-tyrosine kinases and thereby influence the growth and differentiation of the hairy cells.

Rat pituitary N alpha-acetyltransferase.

The rat pituitary contains an enzyme which will acetylate certain ACTH fragments using acetyl coenzyme A. This acetyltransferase activity was found in all three lobes of the rat pituitary as well as in all other tissues examined. The rat pituitary enzyme appears to be largely particulate in nature. The enzyme sedimenting at 27,000 and 100,000 g had specific activities 4-10 times greater than the soluble fraction. The acetyltransferase activity was dependent on substrate concentration (ACTH) and pH, was linear with time, and was inactivated at 55 degrees. The enzyme would acetylate ACTH(1-24), (1-10), and ACTH(4-10), but would not use ACTH(2-10), (3-10), or ACTH(1-8) as substrates. The apparent Km values for the substrates were as follows: AcCoA, 2.2 microM, ACTH(1-24), 4.2 microM; ACTH(1-10), 96 microM; and ACTH(4-10), 37 microM.

Histone H1 kinase in exponential and synchronous populations of Chinese hamster fibroblasts.

Nuclear histone kinase activity, specifically histone H1 phosphotransferase activity, was shown to increase in synchronous Chinese hamster cells from the G1/S boundary to late G2/early M phase. Chromatin extracts purified by DEAE-Sephacel chromatography showed a cAMP-independent kinase activity that demonstrated cell cycle dependence and high specificity for histone H1 as the phosphate acceptor in the presence of [gamma-32P] ATP. This activity was purified approximately 40-fold. Using as substrates calf thymus histone H1 subfractions resolved by Bio-Rex 70 ion exchange chromatography, phosphorylation by the nuclear histone H1 kinase indicated that 32P incorporation into H1-2 was at least twice that for H1-1 and H1-3 subfractions. Both amino- and carboxy-terminal fragments generated by N-bromosuccinimide cleavage were phosphorylated. Phosphoamino acid analysis showed phosphothreonine to be approximately twice as abundant as phosphoserine. Histone H1 kinase activity was not activated by cyclic nucleotides, nor inhibited by cAMP-dependent protein kinase inhibitors or regulatory subunits. There was no effect on activity by Ca2+ alone or in the presence of calmodulin or diacylglycerol. Kinase activity was inhibited by nonhydrolyzable analogs of ATP such as adenyl-5'-yl imidodiphosphate, by 5'-p-fluorosulfonylbenzoyladenosine which binds to the ATP binding site of the enzyme, and by quercetin. Column fractions enriched in histone H1 kinase were labeled with 5'-p-fluorosulfonylbenzoyl[8-14C]adenosine, and peptides were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One band, Mr 67,000, was specifically labeled and may represent the H1 kinase catalytic subunit.

Selective isolation of newly synthesized mammalian mRNA after in vivo labeling with 4-thiouridine or 6-thioguanosine.

Newly synthesized mRNA from hamster cells was labeled in vivo with the thionucleoside analogs 4-thiouridine (4-TU) and 6-thioguanosine (6-TG). The thio-substituted RNA was selectively recovered by Affi-Gel 501 phenylmercury affinity chromatography. Following a 1-h labeling period, enrichment for newly transcribed RNA after a single round of chromatography ranged between 10- and 15-fold when compared with total RNA. Exposure of CHO UrdA- cells, a uridine auxotrophic line, to 50 microM 4-TU allowed for optimal recovery of newly transcribed RNA. Increasing the concentration of 4-TU to 100 microM or labeling with 6-TG at concentrations of 3 microM or greater resulted in similar recoveries from uridine-prototrophic hamster cell lines. For shorter term labeling, exposure of prototrophic cells to 500 microM 4-TU or 100 microM 6-TG for 15 min allowed newly synthesized RNA to be selectively recovered. As a specific test case, enrichment for histone H3.2 mRNA was analyzed after hamster cells were labeled with 4-TU under conditions in which the gene was highly transcriptionally active. Northern blot analysis and the specific activity of thio-substituted RNA revealed a 15-fold enrichment when compared to total RNA. In vivo labeling of cellular RNA with 4-TU or 6-TG should provide a useful method for studying inducible gene expression and for isolating and cloning specific mRNAs from mammalian cells.

Cloning, bacterial expression, purification, and characterization of the cytoplasmic domain of rat LAR, a receptor-like protein tyrosine phosphatase.

This report describes the cloning and characterization of rat leukocyte common antigen-related protein (rLAR), a receptor-like protein tyrosine phosphatase (PTPase). The recombinant cytoplasmic PTPase domain was expressed at high levels in bacteria and purified to homogeneity. Kinetic properties of the PTPase were examined along with potential modulators of PTPase activity. Several sulfhydryl-directed reagents were effective inhibitors, and a surprising distinction between iodoacetate and iodoacetamide was observed. The latter compound was an extremely poor inhibitor when compared to iodoacetate, suggesting that iodoacetate may interact selectively with a positive charge at or near the active site of the enzyme. Site-directed mutants were made at 4 highly conserved cysteine residues found at positions 1434, 1522, 1723, and 1813 within the protein. The Cys-1522/Ser mutation resulted in a 99% loss of enzymatic activity of the pure protein. This observation is consistent with greater than 99% of the PTPase activity being found in the first domain of the PTPase and demonstrates the critical importance of this cysteine residue in catalysis. The recombinant C1522S mutant phosphatase could also be phosphorylated in vitro by protein kinase C and p43v-abl tyrosine kinase. When pure recombinant PTPase was mixed with 32P-labeled tyrosine substrate and then rapidly denatured, a 32P-labeled enzyme intermediate could be trapped and visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The catalytically inactive C1522S mutant did not form the phosphoenzyme intermediate.

Expression and characterization of mutant forms of the type I regulatory subunit of cAMP-dependent protein kinase. The effect of defective cAMP binding on holoenzyme activation.

The mouse wild type and four mutant regulatory type I (RI) subunits were expressed in Escherichia coli and subjected to kinetic analyses. The defective RI subunits had point mutations in either cAMP-binding site A (G200/E), site B (G324/D, R332/H), or in both binding sites. In addition, a truncated form of RI which lacked the entire cAMP-binding site B was generated. All of the mutant RI subunits which bound [3H]cAMP demonstrated more rapid rates of cAMP dissociation compared to the wild type RI subunit. Dissociation profiles showed only a single dissociation component, suggesting that a single nonmutated binding site was functional. The mutant RI subunits associated with purified native catalytic subunit to form chromatographically separable holoenzyme complexes in which catalytic activity was suppressed. Each of these holoenzymes could be activated but showed varying degrees of cAMP responsiveness with apparent Ka values ranging from 40 nM to greater than 5 microM. The extent to which the mutated cAMP-binding sites were defective was also shown by the resistance of the respective holoenzymes to activation by cAMP analogs selective for the mutated binding sites. Kinetic results support the conclusions that 1) Gly-200 of cAMP-binding site A and Gly-324 or Arg-332 of site B are essential to normal conformation and function, 2) activation of type I cAMP-dependent protein kinase requires that only one of the cAMP-binding sites be functional, 3) mutational inactivation of site B (slow exchange) has a much more drastic effect than that of site A on increasing the Ka of the holoenzyme for cAMP, as well as in altering the rate of cAMP dissociation from the remaining site of the free RI subunit. The strong dependence of one cAMP-binding site on the integrity of the other site suggests a tight association between the two sites.

Types I alpha and I beta isozymes of cGMP-dependent protein kinase: alternative mRNA splicing may produce different inhibitory domains.

We recently described a novel isozyme of cGMP-dependent protein kinase (type I beta). It has a structure and peptide substrate specificity which is similar to that of type I alpha, but it has a different cGMP binding behavior, and autophosphorylation occurs almost entirely in serine instead of in both serine and threonine residues (Wolfe, L., Corbin, J.D., and Francis, S.H. (1989) J. Biol. Chem. 264, 7734-7741). An amino-terminal sequence of 31 amino acids derived from three proteolytic fragments of type I beta had 45% homology with a sequence beginning at type I alpha-47. However, sequences of three CNBr peptides of type I beta were identical to sequences of type I alpha beginning at type I alpha-202, -213, and -576 of 11, 27, and 30 residues. These sequences include portions of the catalytic domain and at least one cGMP-binding domain (site 1). Thus, types I alpha and I beta may be produced by alternative splicing of two unique mRNA segments to generate different amino acid sequences in the protein in a region that is amino-terminal to type I alpha-202. This segment in type I beta corresponds to the region in type I alpha that includes the major autophosphorylation site (Thr-58) which is within the domain that is proposed to inhibit catalytic activity. This region presumably interacts with the cGMP-binding site(s) to account for the differences in cGMP-binding behavior between types I alpha and I beta. Even though the sequence of type I beta in the variable region lacks the residue corresponding to Thr-58, it includes a consensus phosphorylation site (KRQAISA) beginning at type I alpha-59, which is absent in type I alpha. The results imply flexibility in the design of the autophosphorylation site and, hence, of the inhibitory domain.

Functional characterization of cAMP-binding mutations in type I protein kinase.

A mutant form of the type I regulatory subunit (RI) of cAMP-dependent protein kinase has been cloned and sequenced (Clegg, C. H., Correll, L. A., Cadd, G. C., and McKnight, G. S. (1987) J. Biol. Chem. 262, 13111-13119) which contains two point mutations in the site B cAMP-binding site, a Gly to Asp at position this report, the effect of each independent mutation on the rate of dissociation of cAMP from RI, the cAMP-mediated activation of holoenzyme and the inducibility of cAMP-responsive genes has been characterized. Dissociation of cAMP from either recombinant wild type RI or the B1 mutant demonstrated biphasic kinetics, indicating two sites with different affinities for cAMP. Dissociation from the B2 subunit, however, was monophasic and very rapid indicating that site B had been destroyed and that the rate of dissociation from site A was increased. The cAMP activation constants (Ka) of the wild type and B1 holoenzymes were 40 and 188 nM, respectively, and demonstrated positive cooperativity, with Hill coefficients of 1.61 for the wild type and 1.67 for B1. The B2 holoenzyme required much greater concentrations of cAMP, 4.7 microM, for half-maximal activation and did not display positive cooperativity. Constitutive expression in mouse AtT20 pituitary cells of the B1 mutant resulted in only a small shift in the Ka for kinase activation in these cells compared with B2 expression which increased the Ka by more than 100-fold. Transient expression of the B1 subunit in human JEG-3 choriocarcinoma cells inhibited forskolin activation of a cAMP-responsive promoter by 35% whereas similar expression of the B2 RI subunit inhibited the response by 90%. These results suggest that the Gly to Asp mutation at amino acid 324 completely blocks cAMP binding to site B whereas the Arg to His mutation at position 332 causes a more subtle alteration in cAMP binding. Expression of either mutant RI in animal cells results in a dominant repression of cAMP-dependent protein kinase activity and cAMP-dependent protein kinase-mediated processes.