PPX, a novel protein serine/threonine phosphatase localized to centrosomes

The amino acid sequence of a novel mammalian protein phosphatase, termed PPX (and designated PPP4 in the human genome nomenclature), has been deduced from the cDNA and shown to be 65% identical to PP2A alpha and PP2A beta and 45% identical to PPI isoforms, the predicted molecular mass being 35 kDa. PPX was expressed in the baculovirus system. Its substrate specificity and sensitivity to the inhibitors, okadaic acid and microcystin, were similar (but not identical) to the catalytic subunit of PP2A. However, PPX did not bind the 65 kDa regulatory subunit of PP2A. The intracellular localization of PPX was investigated by immunofluorescence using two different antibodies raised against bacterially expressed PPX and a PPX-specific peptide. These showed that although PPX was distributed throughout the cytoplasm and the nucleus, intense staining occurred at centrosomes. The centrosomal staining was apparent in interphase and at all stages of mitosis, except telophase. In contrast, antibodies directed against bacterially expressed PP2A were not specifically localized to centrosomes. The human autoantibody #5051, which stains the pericentriolar material, colocalizes with PPX antibodies, suggesting that PPX may play a role in microtubule nucleation.     

DRAKs, novel serine/threonine kinases related to death-associated protein kinase that trigger apoptosis

The present study describes the cloning of two novel serine/threonine kinases termed DRAK1 and DRAK2, whose catalytic domains are related to that of death-associated protein kinase, a serine/threonine kinase involved in apoptosis. Both DRAKs are composed of the N-terminal catalytic domain and the C-terminal domain that is responsible for regulation of kinase activity. DRAK1 and DRAK2 show 59.7% identity and display ubiquitous expression. An in vitro kinase assay revealed that both DRAKs are autophosphorylated and phosphorylate myosin light chain as an exogenous substrate, although the kinase activity of DRAK2 is significantly lower than that of DRAK1. Both DRAKs are exclusively localized to the nucleus. Furthermore, overexpression of both DRAKs induces the morphological changes of apoptosis in NIH 3T3 cells, suggesting the role of DRAKs in apoptotic signaling.     

Identification and characterization of an unusual double serine/threonine protein phosphatase 2C in the malaria parasite Plasmodium falciparum

We have cloned a gene from Plasmodium falciparum with homology to the Mg2+-dependent serine/threonine protein phosphatase 2C (PP2C) family. The predicted coding region is 920 amino acids long, twice the size of other members of this family. We show that this protein can be divided into two halves (Pf2C-1 and Pf2C-2), each a complete phosphatase unit with homology to other phosphatases of this class. To study the function of this PP2C, we have tested the ability of different constructs to complement conditional null mutants of yeast. Our results show that expression of the full-length protein, the first half alone, the second half alone, or a hybrid with the N terminus of the first half and the C terminus of the second half was able to complement the heat shock response defect of a Schizosaccharomyces pombe strain with a PP2C (PTC1) deletion. Recombinant P. falciparum PP2C expressed in Escherichia coli was active in dephosphorylating 32P-labeled casein in an Mg2+- or Mn2+-dependent reaction. Each half alone was also active in recombinant form. Using the two-hybrid system, we have shown that the two halves can interact. Gel filtration assay of P. falciparum protein extracts suggests that full-length PfPP2C is a dimer, and phosphatase activity competition experiments indicate that dimerization of PfPP2C is required for its optimal activity. This unusual phosphatase molecule appears to be composed of four catalytic units on two polypeptide chains.     

PP7, a plant phosphatase representing a novel evolutionary branch of eukaryotic protein Ser/Thr phosphatases

Theoretical quantum mechanical ab initio Hartree-Fock calculations on molecular systems, modeling processes related to the specificity of thymidylate synthase inactivation are reported. We considered several steps of the methylation of the substrate dUMP and 4- or 5-mono- and 4,5-bisubstituted dUMP analogs, as well. The following reactions were modeled: the cysteine residue (Cys198 in the L.casei enzyme) nucleophilic attack on the substrate and the substrate C(5)-H proton abstraction. The substrate was modeled by the 1-methyluracil molecule and its structural analogs. The cysteine Cys198 residue was modeled by the methylmercaptane molecule. The substrate-enzyme binary complex was modeled by the 1-methyl-5,6-dihydro-6-thiomethyl-uracil (P1) molecule. The present theoretical calculations suggest that the cysteine nucleophilic attack on the substrate may result in the SH-group addition to the pyrimidine C(5)=C(6) bond in the course of a weakly exothermic reaction. The formerly presumed enolate carbanion appeared to be weakly stable or unstable and it can readily split into the thiol and pyrimidine residues. The s2-thio- (P2) and s2,4-dithio- (P3) substrate analogs should form stable thiolate anions after cysteine residue attachment to the C(6) position of the pyrimidine ring. Studies of the deformed P1 molecule interacting with a water molecule bound to the pyrimidine C(4)=O carbonyl residue allow a suggestion that this water molecule may be directly involved in the C(5)-H proton abstraction and may serve as a proton transmitter between the substrate and the proton acceptor residue, possibly located on the cofactor N10-nitrogen. Interaction of the pyrimidine C(4)=O group, or its modification, with the N5,10-methylenetetrahydrofolate N(10) nitrogen atom is suggested as an additional factor influencing the inhibition process.     

A serine/threonine protein kinase gene isolated by an in vivo binding procedure using the Arabidopsis floral homeotic gene product, AGAMOUS

During the course of characterizing fragments bound to an Arabidopsis floral protein AGAMOUS in vivo, a gene encoding a putative serine/threonine protein kinase was found on one of the fragments. The deduced 426 amino acid residues of the gene, named APK2a, are 65% identical to a previously reported Arabidopsis serine/threonine protein kinase, APK1a. The gene is composed of 6 exons and maps at 10 cM from the upper end of chromosome 1. Northern hybridization experiments indicated that the gene is strongly expressed in leaves, moderately in roots, and very weakly in flowers. Further in situ analysis of the expression in floral buds showed that the APK2a gene is expressed at pedicels, is not expressed at the floral organ primordia of wild type floral buds, but is moderately expressed in the floral organ primordia of the agamous mutant. In vitro binding assay suggest that the AGAMOUS protein binds to a sequence similar to, but different from, the known MADS-binding consensus sequences, the CArG box, located 3' downstream of the APK2a gene. These results suggest that APK2a expression is negatively regulated by the AG protein. A close homologue of the APK2a gene, named APK2b, was also isolated from the Arabidopsis cDNA library. The expression pattern of the APK2b gene differs from that of APK2a. It is strongly expressed in leaves, moderately in flowers, and weakly in roots.     

Molecular cloning in Arabidopsis thaliana of a new protein phosphatase 2C (PP2C) with homology to ABI1 and ABI2

Trapeziectomy, ligament reconstruction and tendon interposition arthroplasty is one of the most commonly performed procedures to address pain and instability due to osteoarthritis at the basal joint of the thumb. To determine the effect of stress on first metacarpal subsidence, 15 ligament reconstruction and tendon interposition basal joint arthroplasties were evaluated after a mean follow-up of 32 months. Radiographs were obtained of the arthroplasty at rest and then with maximal effort key pinch stress, which is known to subject the first carpometacarpal joint to considerable axial compression stress. Compared with the preoperative x-rays, the first metacarpal had subsided 21% of the arthroplasty space at rest. Under stress, the first metacarpal was found to subside another 10.5% in height. No subluxation of the metacarpal base could be detected. Key pinch strength improved 17% from the preoperative strength. Tip-to-tip pinch strength improved 17% from the preoperative measurement. Grip strength improved 17% from the preoperative measurement. Grip strength was 9% greater than the preoperative grip strength. There was no statistical association between the amount of first metacarpal subsidence and follow-up key pinch, tip pinch, or grip strength. With axial compressive loading of the arthroplasty, such as in lateral pinch, there is some further proximal migration of the first metacarpal, but this is minimal and does not correlate with functional outcome.     

Prk, a cytokine-inducible human protein serine/threonine kinase whose expression appears to be down-regulated in lung carcinomas

We have cloned and characterized a putative protein serine/threonine kinase termed prk through a combination of polymerase chain reaction and conventional cDNA library screening approaches. There are apparently two distinct domains within prk protein deduced from its nucleotide sequences. The amino-terminal portion has the feature of the catalytic domain of a serine/threonine kinase and shows strong homology to mouse fnk and other polo family kinases including mouse snk, human and murine plk, Drosophila polo, and yeast Cdc5. The carboxyl-terminal portion, presumably the regulatory domain, shares extensive homology to mouse fnk. Northern blotting analyses reveal that prk expression is restricted to a very limited number of tissues with placenta, ovaries, and lung containing detectable amounts of prk mRNA. prk mRNA expression is also detected at a low level in the megakaryocytic cell line Dami, MO7e, and three brain glioma cell lines. In addition, refeeding of serum-deprived MO7e, Dami, and K562 cells of hematopoietic origin and GMOO637D of lung fibroblasts rapidly activates prk mRNA expression with its peak induction around 2 h after serum addition. prk gene activation by the serum requires no new protein synthesis. The recombinant cytokines such as interleukin-3 and thrombopoietin also activate prk mRNA expression in MO7e cells. Furthermore, a survey of RNAs isolated from the tumor and the uninvolved tissues from 18 lung cancer patients reveals that prk mRNA expression is significantly down-regulated in tumor tissues. Southern blotting analysis indicates that the prk gene is present in a single copy in the genome of tumors and normal cells. Taken together, these results suggest that prk expression may be restricted to proliferating cells and involved in the regulation of cell cycle progression. The molecular cloning of prk cDNA will facilitate the study of its biological role as well as its potential role in tumorigenesis.     

A novel serine/threonine kinase gene, Gek1, is expressed in meiotic testicular germ cells and primordial germ cells

We have isolated a novel serine/ threonine kinase gene designated Gek1 from mouse primordial germ cell-derived embryonic germ cell. Gek1 is preferentially expressed in meiotic testicular germ cells and primordial germ cells. Gek1 mRNA is also detected in several other tissues, including hematopoietic organs in adult mice and central nervous system in embryos. The Gek1 cDNA encodes a protein with the consensus sequence of the catalytic domain of protein kinases in its N-terminal region. The deduced amino acid sequence of Gek1 in the kinase domain is related to those encoded by the Saccharomyces cerevisiae STE20, CDC15, and Drosophila melanogaster ninaC. The patterns of expression and the structural features of Gek1 suggest that the gene product is involved in signal transduction or nuclear division of germ cells and other proliferating cells. We also show that Gek1 locates on chromosome 11, near the wr locus, showing neuronal and reproductive defects.     

Molecular cloning of SKAP55, a novel protein that associates with the protein tyrosine kinase p59fyn in human T-lymphocytes

In human T-lymphocytes the Src family protein tyrosine kinase p59(fyn) associates with three phosphoproteins of 43, 55, and 85 kDa (pp43, pp55, and pp85). Employing a GST-Fyn-Src homology 2 (SH2) domain fusion protein pp55 was purified from lysates of Jurkat T-cells. Molecular cloning of the pp55 cDNA reveals that the pp55 gene codes for a so far nondescribed polypeptide of 359 amino acids that comprises a pleckstrin homology domain, a C-terminal SH3 domain, as well as several potential tyrosine phosphorylation sites, among which one fulfills the criteria to bind Src-like SH2 domains with high affinity. Consistent with this observation, pp55 selectively binds to isolated SH2 domains of Lck, Lyn, Src, and Fyn but not to the SH2 domains of ZAP70, Syk, Shc, SLP-76, Grb2, phosphatidylinositol 3-kinase, and c-abl in vitro. Based on these properties the protein was termed SKAP55 (src kinase-associated phosphoprotein of 55 kDa). Northern blot analysis shows that SKAP55 mRNA is preferentially expressed in lymphatic tissues. SKAP55 is detected in resting human T-lymphocytes as a constitutively tyrosine phosphorylated protein that selectively interacts with p59(fyn). These data suggest that SKAP55 represents a novel adaptor protein likely involved in Fyn-mediated signaling in human T-lymphocytes.     

Molecular cloning, sequence, and specificity of expression of the gene encoding the low molecular weight human salivary mucin (MUC7)

Previous biochemical studies have determined that human saliva contains high and low molecular weight mucin glycoproteins (MG1 and MG2, respectively) that are structurally distinct. In this study, we describe the isolation and characterization of overlapping cDNA clones which code for the MG2 protein core. DNA sequencing revealed a translated region of 1131 nucleotides encoding a protein of 377 amino acid residues with a molecular mass of 39 kDa. The first 20 N-terminal residues were very hydrophobic and probably comprise the MG2 leader peptide. The region encoding the secreted protein can be divided into three distinct domains; unique 5'- and 3'-translated regions containing 4 and 1 potential N-glycosylation sites, respectively, and a central region of six almost perfect tandem repeats of 23 amino acid residues with a high number of Thr and Ser. No sequence homology with any other human or animal mucins, and no significant homology to any other proteins was found. MG2 mRNA is about 2.5 kilobases long, and its expression appears to be species-, tissue-, and cell-specific. We propose to name this gene MUC7 in accordance with the mucin genes cloned to date named MUC1-MUC6.     

Isolation and characterization of a novel human pancreas-specific gene, pancpin, that is down-regulated in pancreatic cancer cells

We reviewed 19 children and adolescents with cervical spine congenital synostosis as in Klippel-Feil syndrome (KFS), with an average follow-up of 12.5 years. We paid particular attention to neurologic complications associated with cervical spine abnormalities. Five patients were affected by neurologic complications; four underwent a surgical procedure; and 14 had no neurologic finding. Two had hypermobility at one level, and one had hypermobility at two levels. We found that the more numerous the occipito-C1 abnormalities, the more significant the neurologic risk. In contrast, this risk was not related to the number of "mobile blocks" or to age. Various mechanisms of neural complications have been studied in the literature: medullary abnormality, spinal instability, narrowing of the cervical canal, and vascular dysfunction. Surgery is usually thought to be required in cases with neurologic complications. The indication for surgery is, however, less clear in cases of pure instability without neurologic involvement because surgery is likely to increase the future risks at mobile disks either above or below the fuse level. Careful clinical and radiologic observation is necessary in such patients. Magnetic resonance imaging (MRI) with lateral views in flexion and extension seem to be the best method for detecting impingement of the spine on the cord.     

A novel human homologue of the Drosophila frizzled wnt receptor gene binds wingless protein and is in the Williams syndrome deletion at 7q11.23

Prenylated Rab GTPases occur in the cytosol in their GDP-bound conformations bound to a cytosolic protein termed GDP-dissociation inhibitor (GDI). Rab-GDI complexes represent a pool of active, recycling Rab proteins that can deliver Rabs to specific and distinct membrane-bound compartments. Rab delivery to cellular membranes involves release of GDI, and the membrane-associated Rab protein then exchanges its bound GDP for GTP. We report here the identification of a novel, membrane-associated protein factor that can release prenylated Rab proteins from GDI. This GDI-displacement factor (GDF) is not a guanine nucleotide exchange factor because it did not influence the intrinsic rates of nucleotide exchange by Rabs 5, 7 or 9. Rather, GDF caused the release of each of these endosomal Rabs from GDI, permitting them to exchange nucleotide at their intrinsic rates. GDF displayed the greatest catalytic rate enhancement on Rab9-GDI complexes. However, catalytic rate enhancement paralleled the potency of GDI in blocking nucleotide exchange: GDI was shown to be most potent in blocking nucleotide exchange by Rab9. The failure of GDF to act on Rab1-GDI complexes suggests that it may be specific for endosomal Rab proteins. This novel, membrane-associated activity may be part of the machinery used to localize Rabs to their correct intracellular compartments.