The human double-stranded RNA-dependent protein kinase (PKR) is an important component of the interferon response to virus infection. The activation of PKR is accompanied by autophosphorylation at multiple sites, including one in the N-terminal regulatory region (Thr-258) that is required for full kinase activity. Several protein kinases are activated by phosphorylation in the region between kinase subdomains VII and VIII, referred to as the activation loop. We show that Thr-446 and Thr-451 in the PKR activation loop are required in vivo and in vitro for high-level kinase activity. Mutation of either residue to Ala impaired translational control by PKR in yeast cells and COS1 cells and led to tumor formation in mice. These mutations also impaired autophosphorylation and eukaryotic initiation factor 2 subunit ␣ (eIF2␣) phosphorylation by PKR in vitro. Whereas the Ala-446 substitution substantially reduced PKR function, the mutant kinase containing Ala-451 was completely inactive. PKR specifically phosphorylated Thr-446 and Thr-451 in synthetic peptides in vitro, and mass spectrometry analysis of PKR phosphopeptides confirmed that Thr-446 is an autophosphorylation site in vivo. Substitution of Glu-490 in subdomain X of PKR partially restored kinase activity when combined with the Ala-451 mutation. This finding suggests that the interaction between subdomain X and the activation loop, described previously for MAP kinase, is a regulatory feature conserved in PKR. We found that the yeast eIF2␣ kinase GCN2 autophosphorylates at Thr-882 and Thr-887, located in the activation loop at exactly the same positions as Thr-446 and Thr-451 in PKR. Thr-887 was more critically required than was Thr-882 for GCN2 kinase activity, paralleling the relative importance of Thr-446 and Thr-451 in PKR. These results indicate striking similarities between GCN2 and PKR in the importance of autophosphorylation and the conserved Thr residues in the activation loop.

We report a fast, sensitive, and robust procedure for the identification of precise phosphorylation sites in proteins separated by polyacrylamide gel electrophoresis by a combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF) and online capillary liquid chromatography electrospray tandem ion trap mass spectrometry (LC/ESI/MS/MS). With this procedure, a single phosphorylation site was identified on as little as 20 ng (500 fmol) of the baculovirus-expressed catalytic domain of myosin I heavy-chain kinase separated by gel electrophoresis. The phosphoprotein is digested in the gel with trypsin, and the resulting peptides are extracted with > 60% yield and analyzed by MALDI/TOF before and after digestion with a phosphatase to identify the phosphopeptides. The phosphopeptides are then separated and fragmented in an on-line LC/ESI ion trap mass spectrometer to identify the precise phosphorylation sites. This procedure eliminates any off-line HPLC separation and minimizes sample handling. The use of MALDI/TOF and LCQ, two types of mass spectrometers that are widely available to the biological community, will make this procedure readily accessible to biologists. We applied this technique to identify two autophosphorylation sites and to assign at least another 12 phosphorylation sites to two tryptic peptides in a series of experiments using a gel slice containing only 200 ng (3 pmol) of human double-stranded RNA-activated protein kinase expressed in a mutant strain of the yeast Saccharomyces cerevisiae.

De novo peptide sequencing in an ion trap mass spectrometer coupled on-line with a capillary HPLC using 18O labeling provides a viable alternative to the method using the combination of nanospray, 18O labeling and a quadrupole/time-of-flight mass spectrometer. Seven to sixteen amino acid residues can be sequenced from the liquid chromatography/randem mass spectrometry (LC/MS/MS) spectra. This approach combines the benefit of capillary LC and the high sensitivity of the ion trap operated in the MS/MS mode. The wide availability of the LCQ mass spectrometer makes this approach readily adaptable to the biological mass spectrometry community.

Xenotransplantation may bridge the widening gap between the shortage of donor organs and the increasing number of patients waiting for transplantation. However, a major safety issue is the potential cross-species transmission of porcine endogenous retroviruses (PERV). This problem could be resolved if it is possible to produce pigs that do not contain replication-competent copies of this virus. In order to determine the feasibility of this, we have determined the number of potentially replication-competent full-length PERV proviruses and obtained data on their integration sites within the porcine genome. We have screened genomic DNA libraries from a Large White pig for potentially intact proviruses. We identified six unique PERV B proviruses that were apparently intact in all three genes, while the majority of isolated proviruses were defective in one or more genes. No intact PERV A proviruses were found in this pig, despite the identification of multiple defective A proviruses. Genotyping of 30 unrelated pigs for these unique proviruses showed a heterogeneous distribution. Two proviruses were uncommon, present in 7 of 30 and 3 of 30 pigs, while three were each present in 24 of 30 pigs, and one was present in 30 of 30 animals examined. Our data indicate that few PERV proviruses in Large White pigs are capable of productive infection and suggest that many could be removed by selective breeding. Further studies are required to determine if all potentially functional proviruses could be removed by breeding or whether gene knockout techniques will be required to remove the residuum.

Biochemistry. In the article ''Identification by mass spectrometry of the phosphorylated residue responsible for activation of the catalytic domain of myosin I heavy chain kinase, a member of the PAK͞STE20 family'' by Joanna Szczepanowska, Xiaolong Zhang, Christopher J. Herring, Jun Qin, Edward D. Korn, and Hanna Brzeska, which appeared in number 16, August 5, 1997, of Proc. Natl. Acad. Sci. USA (94,(8503)(8504)(8505)(8506)(8507)(8508), the authors wish to note that in Fig. 3, the ions of m͞z 1345.3 and 1247.1 were incorrectly identified as b 13 and b 13 ⌬ , respectively, produced by cleavage of the peptide AS(Pi)VVGTTYW-MAPEVVK between E and V (Fig. 3 Inset). In fact, these ions are b 12 and b 12 ⌬ , produced by cleavage of the peptide between P and E. This correction has no effect on the conclusion that the phosphorylated residue is serine. Also, in Table 2, reference numbers 22-24 should be 21-23 (the reference in the legend is cited correctly) and the MIHCK sequence is from residue 624 to residue 638.Cell Biology. In the article ''Subtraction hybridization identifies a transformation progression-associated gene PEG-3 with sequence homology to a growth arrest and DNA damageinducible gene'' by Zao

A unique preconcentrator was constructed for on-line preconcentration using capillary electrophoresis generating concentration detection limits for peptides in the mid-picomolar range. Several common sheathless electrospray configurations were constructed in conjunction with the preconcentrator and were compared. It was found that a palladium wire interface was the most reliable interface and the problems encountered using this interface are discussed. A tryptic digest of the myosin I heavy chain kinase that contains two phosphopeptides was separated by capillary electrophoresis using this interface and the on-line preconcentrator.

Phosphorylation of Ser-627 is both necessary and sufficient for full activity of the expressed 35-kDa catalytic domain of myosin I heavy chain kinase (MIHCK). Ser-627 lies in the variable loop between highly conserved residues DFG and APE at a position at which a phosphorylated Ser͞Thr also occurs in many other Ser͞Thr protein kinases. The variable loop of MIHCK contains two other hydroxyamino acids: Thr-631, which is conserved in almost all Ser͞Thr kinases, and Thr-632, which is not conserved. We determined the effects on the kinase activity of the expressed catalytic domain of mutating Ser-627, Thr-631, and Thr-632 individually to Ala, Asp, and Glu. The S627A mutant was substantially less active than wild type (wt), with a lower k cat and higher K m for both peptide substrate and ATP, but was more active than unphosphorylated wt. The S627D and S627E mutants were also less active than phosphorylated wt, i.e., acidic amino acids cannot substitute for phospho-Ser-627. The activity of the T631A mutant was as low as that of the S627A mutant, whereas the T632A mutant was as active as phosphorylated wt, indicating that highly conserved Thr-631, although not phosphorylated, is essential for catalytic activity. Asp and Glu substitutions for Thr-631 and Thr-632 were inhibitory to various degrees. Molecular modeling indicated that Thr-631 can hydrogen bond with conserved residue Asp-591 in the catalytic loop and that similar interactions are possible for other kinases whose activities also are regulated by phosphorylation in the variable loop. Thus, this conserved Thr residue may be essential for the activities of other Ser͞Thr protein kinases as well as for the activity of MIHCK.

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