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R Jakobi

Publications and source records attributed to R Jakobi.

16 recordsLinked to original sources
PubMed1996

Molecular cloning and sequencing of the cytostatic G protein-activated protein kinase PAK I.

The serine/threonine protein kinase PAK I (p2l-activated protein kinase), a ubiquitous multipotential protein kinase of 58-60 kDa, has been shown to have cytostatic properties. Data from our laboratory show that PAK I is highly active in oocytes and quiescent and serum-starved cells, and injection of active PAK I into one blastomere of two-cell frog embryos inhibits cleavage of the injected blastomere. To clone the cDNA encoding PAK I, purified peptides from rabbit PAK I were sequenced, degenerate oligonucleotides were used to isolate PAK I clones from a rabbit spleen library, and the 5'-terminus was obtained by polymerase chain reaction. The entire cDNA sequence extends over 4471 nucleotides, with an open reading frame for a protein of 524 residues and a 3'-noncoding region of 2826 nucleotides. Clones with the same open reading frame but with 3'-noncoding regions of 1055 and 2478 nucleotides were isolated, suggesting the generation of different transcripts by alternative termination of transcription. The amino acid sequence of PAK I shows high homology to the p2l-activated protein kinases from human placenta and rat brain and to yeast STE20. PAK I is activated by Cdc42(GTP). The PAK enzymes have been proposed to regulate the stress-activated protein kinase (also known as the Jun kinase) signaling pathway (Coso, O. A., Chiariello, M., Yu, J.-C., Teramoto, H., Crespo, P., Xu, N., Miki, T., and Gutkind, J. S. (1995) Cell 81, 1137-1146; Minden, A., Lin, A., Claret, F.-X., Abo, A., and Karin, M. (1995) Cell 81, 1147-1157).

Amino Acid Sequence↗
PubMed1995

Site-directed mutagenesis and structure/function studies of casein kinase II correlate stimulation of activity by the beta subunit with changes in conformation and ATP/GTP utilization.

Casein kinase II exists in vivo as an active holoenzyme consisting of catalytic alpha and/or alpha' and regulatory beta subunits, which form a tetrameric structure of alpha 2 beta 2. Unlike most other protein kinases, casein kinase II uses both ATP and GTP effectively as phosphate donors. Two residues unique to the catalytic subunit of casein kinase II, Val66 and Trp176, were mutated to Ala66 and Phe176, respectively, the amino acids present in more than 95% of the identified protein kinase sequences. Using recombinant alpha subunit expressed in Escherichia coli, the mutations have been previously shown to reduce the utilization of GTP by changing Km values for ATP and GTP and to reduce the approximate fivefold stimulation observed upon addition of the regulatory subunit [Jakobi, R. & Traugh, J. A. (1992) J. Biol. Chem. 267, 23,894-23,902]. In this study, the mutations are shown to affect the catalytic activity of the reconstituted holoenzyme by changing both Km and Vmax values. The Vmax for ATP is reduced by the mutation of Trp176 to phenylalanine, but no change is observed with GTP. The Val66 to alanine and Val66/Trp176 to alanine/phenylalanine mutations reduce the Vmax values for ATP and GTP to levels comparable to those of the catalytic subunits alone, indicating that changes in the stimulation of activity by the beta subunit are due to changes in Vmax. Structural studies using ultraviolet CD spectroscopy show that changes in stimulation of Vmax by the beta subunit are correlated with changes in the secondary structure; the extent of these changes is reduced by both mutations. Correlation of changes in secondary structure and stimulation of activity by the beta subunit indicate that the formation of the wild-type holoenzyme causes conformational changes in the active site, leading to an increased rate of reaction. As shown by the mutations, Val66 and Trp176 are involved both in the conformational changes and in the selectivity of ATP and GTP.

Adenosine Triphosphate↗
PubMed1995

Analysis of the ATP/GTP binding site of casein kinase II by site-directed mutagenesis.

Casein kinase II is one of only a few protein kinases which effectively utilize ATP and GTP in the phosphotransferase reaction. Two residues conserved in the ATP binding domain of other protein kinases are unique to the catalytic (alpha) subunit of casein kinase II. Val-66 is present in subdomain II and Trp-176 in subdomain VII, while > 95% of the other protein kinases contain alanine and phenylalanine, respectively. The residues in the alpha subunit of casein kinase II were changed to the conserved residues via single and double mutations by site-directed mutagenesis. These mutations enhanced the utilization of ATP over GTP by altering the K(m) values of the alpha subunit for ATP and GTP. Following reconstitution of the catalytic subunit with the regulatory (beta) subunit, both the K(m) and Vmax values of the reconstituted alpha 2 beta 2 holoenzyme were altered. Interestingly, the mutations also reduced or eliminated the 4- to 5-fold increase in catalytic activity observed with the holoenzyme over that of the alpha subunit alone. This was due to changes in secondary structure of the holoenzyme as shown by UV circular dichroism spectroscopy. Taken together, the data indicate that utilization of both ATP and GTP can be directly correlated with stimulation of catalytic activity by the regulatory subunit and suggest a co-evolution of these separate functions.

Adenosine Triphosphate↗
PubMed1994

Reconstitution of heterologous and chimeric casein kinase II with recombinant subunits from human and Drosophila: identification of species-specific differences in the beta subunit.

Casein kinase II is composed of two catalytic (alpha) and two regulatory (beta) subunits, the amino acid sequences of the alpha and beta subunits are highly conserved between species. To examine whether heterologous casein kinase II could be formed, recombinant alpha and beta subunits from human and Drosophila were reconstituted from inclusion bodies. Casein kinase II containing either human alpha and Drosophila beta or Drosophila alpha and human beta subunits exhibited enzymatic properties similar to those of the homologous holoenzymes with regard to specific activity, salt optima, and autophosphorylation. However, renaturation and reconstitution of casein kinase II was dependent on the type of beta subunits and the redox conditions, with the Drosophila beta subunits requiring more reduced conditions. Chimeric beta subunits prepared from human and Drosophila cDNA revealed that the N-terminal region was responsible for the requirement for the reduced redox state during renaturation. The N-terminal region also affected solubility and electrophoretic mobility of the beta subunit.

Amino Acid Sequence↗
PubMed1994

Modes of regulation of casein kinase II.

Casein kinase II is unique when compared to other protein kinases; it utilizes GTP with almost the same effectiveness as ATP and exists as an active holoenzyme which does not need to be activated by dissociation of regulatory subunits or unfolding of regulatory domains. In vitro, the activity of casein kinase II is inhibited by acidic compounds and stimulated by basic compounds. Casein kinase II activity is inhibited by 2,3-bisphosphoglycerate and stimulated by polyamines at levels which are physiological in red cells. To examine the effects of autophosphorylation of the beta subunit on activity, two mutants of the Drosophila beta subunit have been constructed in which Ser-4 or Ser-(2-4) are changed to alanine residues. Analysis of autophosphorylation with wild-type and mutant recombinant holoenzymes reveals Ser-2 and Ser-3 as the major autophosphorylation sites. Autophosphorylation does not affect the phosphorylation of casein, but reduces the rate of phosphorylation of glycogen synthase by 30%, elongation factor I by 50-70%, and calmodulin by 20-40%. The data indicate that autophosphorylation of the beta subunit can negatively regulate the phosphotransferase activity of casein kinase II with physiological substrates. To examine regulation of casein kinase II activity by the beta subunit, recombinant alpha and beta subunits from human and Drosophila were expressed in Escherichia coli. Upon formation of the holoenzyme, the beta subunit stimulated the catalytic activity 4- to 5-fold. The catalytic alpha subunit contains the eleven conserved subdomains characteristic of all protein kinases.(ABSTRACT TRUNCATED AT 250 WORDS)

2,3-Diphosphoglycerate↗
PubMed1993

[Orthotopic liver transplantation with arterial re-anastomosis in the rat--a detailed description of technique and analysis of possible errors].

Liver transplantation (LTX) in the rat is a frequently used transplantation model. In the present study we give a detailed description of the suture technique for all anastomoses, except the bile duct, with arterialization of the transplanted liver over an aortoceliac segment to the recipient's aorta. Using this technique, we finally achieved survival rates of 80%. During the development stages, the main cause of death was an exceedingly long anhepatic time (> 20 min); later on, complications occurred only from insufficient anastomoses. The advantage of the suture technique over the cuff technique is that a more physiological anastomosis is obtained with less trauma to the recipient when using rearterialization. Despite the complicated suture technique, the anhepatic time can be well reduced to below 20 min.

Anastomosis, Surgical↗
PubMed1992

Characterization of the phosphotransferase domain of casein kinase II by site-directed mutagenesis and expression in Escherichia coli.

The catalytic alpha subunit of casein kinase II contains the 11 conserved domains characteristic of all protein kinases. Domain II and VII are involved in nucleotide binding and phosphotransfer. Two residues of the alpha subunit, Val-66 (in domain II) and Trp-176 (in domain VII), were changed to Ala-66 and Phe-176, the residues present in more than 95% of the identified protein kinase sequences. These changes altered the selectivity of the alpha subunit for ATP and GTP. The Ala-66 mutant showed an increase in the Km value for GTP from 45 to 71 microM, while the Km value for ATP decreased from 13 to 9 microM. The Km value for ATP with the Phe-176 mutant showed a decrease from 13 to 7 microM. A double mutant of Ala-66/Phe-176 showed the combined effects, with a Km of 6 microM for ATP and 70 microM for GTP. Alteration of Trp-176 to Lys-176, an amino acid which is not present in the corresponding position of any known protein kinase, resulted in a lack of phosphotransferase activity. The mutations, Val-66 to Ala-66 and Trp-176 to Phe-176, also altered the interaction of the alpha subunit with the regulatory beta subunit. In contrast to the wild-type alpha subunit, which was stimulated 4-fold by addition of the beta subunit, the Ala-66 and Ala-66/Phe-176 mutants were not stimulated by the beta subunit, while the Phe-176 mutant was stimulated only 2.5-fold. All of the reconstituted holoenzymes were similar in molecular weight to the native holoenzyme. The stimulation of the phosphotransferase activity toward beta-casein B by spermine and polylysine, which is mediated by the beta subunit, was similar for holoenzymes reconstituted with either wild-type or mutant alpha subunits. Therefore, binding of the beta subunit appears to alter the active site of the alpha subunit directly or indirectly by inducing a conformational change. Ala-66 and Phe-176 mutations appear to change the structure of the alpha subunit sufficiently so that interaction of the subunits is altered and the stimulatory effect of the beta subunit is reduced or eliminated.

Amino Acid Sequence↗
PubMed1992

[Proliferation kinetic studies in the syngeneic liver transplantation mode in the rat. Effect of re-arterialization on the transplant].

Successful orthotopic liver transplantation (OLT) can be achieved in the rat. We used bromo-2-deoxyuridine (BrdU) as a proliferation marker to document morphological differences between OLT with and without rearterialization. Animals with portal anastomosis alone had a significantly increased proliferation rate of hepatocytes, Kupffer cells, and bile duct epithelial cells, as indicated by strong staining with BrdU, 8 days post-transplant compared to animals with rearterialization. Regeneration of ischemically damaged liver parenchymal cells may account for this observation. Thus, OLT with rearterialization appears to be the more physiological transplant model.

Anastomosis, Surgical↗
PubMed1992

[In-vitro activity pattern of gallbladder muscle tissues in patients with and without gallstones--variable response to cholecystokinin, motilin and neurotensin].

Data about gallbladder motility in stone disease and stone free conditions are discussed controversially. This study is aimed at to evaluate in vitro the responses of human gallbladder muscle stripes to cholecystokinin, motilin and neurotensin. Following the results, a stratification of the "stone-disease-group" seems to be necessary: 41% of the gallbladders (stone disease) demonstrate a deep reduction in contractility to cholecystokinin ("non-contractors"), whereas 59% of the gallbladders contract in the same pattern like the controls ("contractors"). Interestingly, the majority of the gallbladders in the "contractor" group demonstrate a high spontaneous activity which is absent in stone free gallbladders. Motilin and neurotensin either do not induce any effect.

Adult↗
PubMed1991

Structure of the gene encoding human casein kinase II subunit beta.

Casein kinase II (CKII) is a ubiquitous serine/threonine protein kinase with numerous key functions in cell metabolism and growth. The human CKII has a tetrameric structure; two catalytic subunits (alpha and alpha') form the holoenzyme together with two presumably regulatory subunits (beta). The gene encoding CKII subunit beta was isolated from human genomic DNA and analyzed for its primary structure using exclusively nonradioactive procedures. The gene was found to span 4.2 kilobase pairs and to be composed of seven exons. Exon sizes range from 76 (exon 5) to 329 base pairs (bp) (exon 1), intron sizes from 145 (intron V) to 965 bp (intron II). All exon-intron junctional sequences conform to the canonical GT-AG rule. Primer extension analysis determined three transcription initiation sites, at 951, 919, and (minor) 840 bp upstream of the translation start site. The translation start is located early in the second exon; exon 1 is untranslated. The 3'-cleavage/polyadenylation signal sequence (AA-TAAA) is in the last exon at position 4173 bp relative to the first transcription initiation site. The coding sequence for CKII beta comprises 648 nucleotides identical to the published CKII beta-cDNA sequence (Jakobi, R., Voss, H., and Pyerin, W. (1989) Eur. J. Biochem. 183, 227-233). The upstream promoter region of the CKII beta gene contains multiple potential gene regulatory sequence elements, noticeable DNA structures, and the characteristics of a housekeeping gene (more than one transcription initiation site, lack of a TATA-box, presence of a CpG island, occurrence of multiple GC boxes and of nonstandard positioned CCAAT boxes). The CKII beta gene promoter shares common features with that of mammalian protein kinases and is closely related to the regulatory subunit gene promoter of cAMP-dependent protein kinase.

Amino Acid Sequence↗
PubMed1991

Cell growth stimulation by EGF: inhibition through antisense-oligodeoxynucleotides demonstrates important role of casein kinase II.

Casein kinase II (CKII) is a highly conserved ubiquitous serine/threonine kinase composed of two catalytically active subunits (alpha and/or alpha') and two presumably regulatory subunits (beta). CKII has numerous cellular functions including a possible role in mitogenic signaling. To address this question, growth-arrested primary human fibroblasts (IMR-90) were exposed prior growth stimulation by epidermal growth factor (EGF) to oligodeoxynucleotides complementary to the translation start region of mRNAs coding for CKII alpha and beta subunits. A significant inhibition of growth stimulation (up to 60%) was observed with both antisense-alpha and antisense-beta. The inhibition was reversible, became decreased with mutated antisense-oligodeoxynucleotides, and neutralized by simultaneous presence of respective sense-oligodeoxynucleotides. The expected down-regulation of CKII protein due to hybrid formation of antisense-oligodeoxynucleotides with target mRNAs was investigated by determination of the intracellular protein level of CKII beta-subunit by immunofluorescence and quantitative image analysis. The protein was revealed to be localized predominantly in the nucleus and to become significantly decreased due to antisense-beta treatment of cells. The maximum decrease coincided with the early phase (first several hours) of growth stimulation by EGF when antisense-beta incubation was started 6-2 h before growth stimulation, the period within which application of antisense-alpha and antisense-beta caused the maximum of inhibition of growth stimulation. Thus CKII obviously plays, with both subunit alpha and subunit beta, an important role in the early phase of mitogenic stimulation.

Base Sequence↗
PubMed1989

Human phosvitin/casein kinase type II. Molecular cloning and sequencing of full-length cDNA encoding subunit beta.

Phosvitin/casein kinase type II is an ubiquitous, highly conserved enzyme consisting of subunits alpha, alpha' and beta. Subunit beta, presumably serving regulatory functions, was prepared from human placenta and the amino acid sequence of a protease digestion peptide was determined. The deduced nucleotide sequence was employed for the synthesis of a mixture of 20mers as a hybridization probe to screen a lambda gt10 HeLa cell cDNA library for clones encoding subunit beta. A full-length clone consisting of 1013 bp was isolated and the sequence of both strands determined. The deduced amino acid sequence of the largest open reading frame encodes 215 amino acid residues predicting a maximum Mr of 24925. The nucleotide sequence of the human beta subunit shows a similarity of 75% to that of Drosophila melanogaster and the deduced amino acid sequence a similarity of 88% and 97% to that of D. melanogaster and bovine lung, respectively. No further protein sequence currently known exhibits any notable similarity. Using a 537-bp restriction fragment of the cDNA clone representing 80% of the coding region, a 1-kb subunit-beta transcript was detected by Northern hybridization in total RNA prepared from human epithelial cells and placenta as well as from bovine heart suggesting a single transcript of the beta-subunit gene and also demonstrating high similarity also between the human and bovine nucleotide sequences. The same restriction fragment was used as the probe to indicate in Southern hybridizations that the corresponding genomic DNA contains at least one intron of roughly 2.5 kb in length and presumably is a single copy gene.

Amino Acid Sequence↗
PubMed1988

Filter-supported preparation of lambda phage DNA.

A rapid and simple method is described for the isolation of DNA from phage lambda which requires neither special equipment nor expensive material such as cesium chloride for ultracentrifugation nor extractions with organic solvents or ethanol precipitation. Microgram quantities of lambda DNA are obtained in less than 2 h from 90-mm plate lysates or 5-ml liquid cultures. The method allows the simultaneous isolation of large numbers of probes, e.g., clones from phage libraries. Lambda phages are precipitated by polyethylene glycol/sodium chloride and recovered by low speed centrifugation onto glass fiber filters positioned in disposable syringes. The DNA of phages is released by a 50% formamide/4 M sodium perchlorate solution, washed in filter-bound form, eluted with a small volume of low-salt buffer or water, and finally recovered by centrifugation. Comparison of the DNA isolated by this method with that obtained by two conventional procedures reveals both a similar recovery and a similar suitability for restriction enzyme digestion and subcloning.

Bacteriophage lambda↗