Radiation inactivation and in situ renaturation of protein tyrosine kinases reveal a major 50-kDa enzyme as part of a membrane complex present in dividing but not in resting prostatic epithelial cells

1996 ◽  
Vol 74 (1) ◽  
pp. 75-85 ◽  
Author(s):  
Linh The Nguyen ◽  
Yves Durocher ◽  
Guy Beauregard ◽  
Sylvain Tessier ◽  
Azeddine Atfi ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Specific Activity ◽  
Protein Tyrosine Kinases ◽  
Radiation Inactivation ◽  
Protein Tyrosine ◽  
Dividing Cells ◽  
Membrane Bound

Because protein tyrosine kinases play a crucial role in the regulation of cell division and carcinogenesis, we have herein measured such enzyme activities (specific activity and subcellular distribution) and compared their characteristics with respect to hydrodynamic properties and radiation inactivation sizes as well as renaturation after electrophoresis in denaturing conditions in canine prostatic epithelial cells either in a resting (freshly isolated) or in a dividing (cultured cells) state. In quiescent cells, most protein tyrosine kinase activity was expressed by soluble proteins with a Stokes' radius (Rs) of 3.05 nm, a sedimentation coefficient (S20,w) of 4.0 S, and a molecular mass of 50 kDa. By contrast, in dividing cells (three days in primary culture), the specific activity was higher and the enzyme was mainly membrane bound. The use of a detergent (Triton X-100) allowed the extraction of most of that enzyme; its partial specific volume, S20,w, and Rs were then 0.883 cm3/g, 4.0 S, and 5.6 nm, respectively, hence yielding a molecular mass of 215 kDa, which decreased to 125–145 kDa when corrected for detergent binding. Probing these chromatography-peak fractions, 50 kDa from cytosol of resting cells and 215 kDa from membrane extracts of dividing cells, with a phosphotyrosine antibody following their incubation with ATP and electrophoresis in denaturing conditions revealed the presence of a common 50-kDa phosphotyrosylated protein along with three other bands (130, 75, and 40 kDa) in the high-Mr peak of enzyme. However, the radiation inactivation size for protein tyrosine kinases expressed in both resting and dividing cells were similar, 47.2 ± 8.7 and 44.5 ± 6.1 kDa, respectively. Furthermore, by renaturation after electrophoresis in denaturing conditions, major protein tyrosine kinase polypeptides of 50 kDa were identified in both cell populations. Taken together, these results indicate that, in dividing prostatic epithelial cells, membrane-bound protein tyrosine kinases of low molecular weight with properties similar to those of monomeric soluble forms present in quiescent cells are part of high-molecular weight complexes. This activation process may be critical for hormone-independent proliferation of prostatic epithelial cells.Key words: protein tyrosine kinase, kinase renaturation, cell division, prostate, radiation inactivation.

scholarly journals Novel yeast protein kinase (YPK1 gene product) is a 40-kilodalton phosphotyrosyl protein associated with protein-tyrosine kinase activity.

1990 ◽  
Vol 10 (12) ◽  
pp. 6244-6256 ◽  
Author(s):  
D Dailey ◽  
G L Schieven ◽  
M Y Lim ◽  
H Marquardt ◽  
T Gilmore ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Protein Tyrosine Kinases ◽  
Tyrosine Kinase Activity ◽  
Protein Tyrosine ◽  
Protein Tyrosine Kinase Activity

Extracts of bakers' yeast (Saccharomyces cerevisiae) contain protein-tyrosine kinase activity that can be detected with a synthetic Glu-Tyr copolymer as substrate (G. Schieven, J. Thorner, and G.S. Martin, Science 231:390-393, 1986). By using this assay in conjunction with ion-exchange and affinity chromatography, a soluble tyrosine kinase activity was purified over 8,000-fold from yeast extracts. The purified activity did not utilize typical substrates for mammalian protein-tyrosine kinases (enolase, casein, and histones). The level of tyrosine kinase activity at all steps of each preparation correlated with the content of a 40-kDa protein (p40). Upon incubation of the most highly purified fractions with Mn-ATP or Mg-ATP, p40 was the only protein phosphorylated on tyrosine. Immunoblotting of purified p40 or total yeast extracts with antiphosphotyrosine antibodies and phosphoamino acid analysis of 32P-labeled yeast proteins fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the 40-kDa protein is normally phosphorylated at tyrosine in vivo. 32P-labeled p40 immunoprecipitated from extracts of metabolically labeled cells by affinity-purified anti-p40 antibodies contained both phosphoserine and phosphotyrosine. The gene encoding p40 (YPK1) was cloned from a yeast genomic library by using oligonucleotide probes designed on the basis of the sequence of purified peptides. As deduced from the nucleotide sequence of YPK1, p40 is homologous to known protein kinases, with features that resemble known protein-serine kinases more than known protein-tyrosine kinases. Thus, p40 is a protein kinase which is phosphorylated in vivo and in vitro at both tyrosine and serine residues; it may be a novel type of autophosphorylating tyrosine kinase, a bifunctional (serine/tyrosine-specific) protein kinase, or a serine kinase that is a substrate for an associated tyrosine kinase.

Novel yeast protein kinase (YPK1 gene product) is a 40-kilodalton phosphotyrosyl protein associated with protein-tyrosine kinase activity

1990 ◽  
Vol 10 (12) ◽  
pp. 6244-6256
Author(s):  
D Dailey ◽  
G L Schieven ◽  
M Y Lim ◽  
H Marquardt ◽  
T Gilmore ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Protein Tyrosine Kinases ◽  
Tyrosine Kinase Activity ◽  
Protein Tyrosine ◽  
Protein Tyrosine Kinase Activity

Extracts of bakers' yeast (Saccharomyces cerevisiae) contain protein-tyrosine kinase activity that can be detected with a synthetic Glu-Tyr copolymer as substrate (G. Schieven, J. Thorner, and G.S. Martin, Science 231:390-393, 1986). By using this assay in conjunction with ion-exchange and affinity chromatography, a soluble tyrosine kinase activity was purified over 8,000-fold from yeast extracts. The purified activity did not utilize typical substrates for mammalian protein-tyrosine kinases (enolase, casein, and histones). The level of tyrosine kinase activity at all steps of each preparation correlated with the content of a 40-kDa protein (p40). Upon incubation of the most highly purified fractions with Mn-ATP or Mg-ATP, p40 was the only protein phosphorylated on tyrosine. Immunoblotting of purified p40 or total yeast extracts with antiphosphotyrosine antibodies and phosphoamino acid analysis of 32P-labeled yeast proteins fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the 40-kDa protein is normally phosphorylated at tyrosine in vivo. 32P-labeled p40 immunoprecipitated from extracts of metabolically labeled cells by affinity-purified anti-p40 antibodies contained both phosphoserine and phosphotyrosine. The gene encoding p40 (YPK1) was cloned from a yeast genomic library by using oligonucleotide probes designed on the basis of the sequence of purified peptides. As deduced from the nucleotide sequence of YPK1, p40 is homologous to known protein kinases, with features that resemble known protein-serine kinases more than known protein-tyrosine kinases. Thus, p40 is a protein kinase which is phosphorylated in vivo and in vitro at both tyrosine and serine residues; it may be a novel type of autophosphorylating tyrosine kinase, a bifunctional (serine/tyrosine-specific) protein kinase, or a serine kinase that is a substrate for an associated tyrosine kinase.

scholarly journals Cytosolic Protein-Tyrosine Kinase Activities in Various Rat Tissues

1989 ◽  
Vol 26 (2) ◽  
pp. 164-168 ◽  
Author(s):  
Tomoko Kobayashi ◽  
Shun-Ichi Nakamura ◽  
Hirohei Yamamura
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Total Activity ◽  
Protein Tyrosine Kinases ◽  
Rat Tissues ◽  
Protein Tyrosine ◽  
Cytosolic Protein ◽  
Lymphatic Organs ◽  
Assay Conditions

Suitable assay conditions for the detection of cytosolic protein-tyrosine kinase activities in crude extracts of various rat tissues have been determined. Cytosolic protein-tyrosine kinases showed common characteristics including substrate specificity and divalent cation requirement. Using (Val5) angiotensin II and Mn2+ rather than a src-related synthetic peptide, E11G1, and Mg2+, we obtained higher activities of cytosolic protein-tyrosine kinases. Among various rat tissues tested, spleen, bone marrow, thymus, small intestine, appendix and lung, in decreasing order of total activity, contained high activities of cytosolic protein-tyrosine kinases. These results suggest that the enzyme activities in lymphatic organs and in organs closely related to cell proliferation are high. The assay system described allows the precise measurement of cytosolic protein-tyrosine kinase activity in various rat tissues, both normal and malignant.

scholarly journals Differential modulation of plasminogen activator gene expression by oncogene-encoded protein tyrosine kinases.

1993 ◽  
Vol 13 (9) ◽  
pp. 5888-5897 ◽  
Author(s):  
S M Bell ◽  
D C Connolly ◽  
N J Maihle ◽  
J L Degen
Keyword(s):  
Gene Expression ◽  
Protein Kinase ◽  
Tyrosine Kinase ◽  
Plasminogen Activator ◽  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Protein Tyrosine Kinases ◽  
Mrna Levels ◽  
Protein Tyrosine ◽  
Upa Mrna

Urokinase-type plasminogen activator (uPA) gene transcription is increased > or = 50-fold in chicken embryo fibroblasts (CEF) following transformation by the protein tyrosine kinase pp60v-src. Protein phosphorylation appears to play a critical role in uPA gene expression in these cells; protein kinase C-activating phorbol esters cooperate with pp60v-src to synergistically increase uPA mRNA, whereas cyclic AMP (cAMP)-dependent protein kinase-activating agents (e.g., 8-bromo cAMP) repress uPA mRNA levels. To explore the relationship between transforming oncogenes and uPA gene expression, uPA mRNA levels were measured in CEF infected with selected avian retroviruses. We report that v-ras and the transforming protein tyrosine kinases v-src, v-yes, and v-ros all increase cellular uPA mRNAs. However, transformation with the protein tyrosine kinase encoded by v-erbB, or the nuclear proteins encoded by v-jun, v-ski, or v-myc, did not increase uPA mRNA detectably. Ras and all of the protein tyrosine kinases analyzed, including the v-erbB product, but none of the nuclear oncoproteins sensitized cells to phorbol ester induction of uPA gene expression. Thus, increased uPA gene expression is not simply a secondary consequence of cell transformation but, rather, is regulated or comodulated by only a subset of oncogene products. Analysis of cells expressing site-directed mutants of pp60v-src showed that the induction of the uPA gene is dependent on protein tyrosine kinase catalytic activity, myristylation, and plasma membrane localization. However, these properties together are not sufficient; an additional feature in the src homology 2 domain is also required. The major sites of serine phosphorylation, serines 12 and 17, and the autophosphorylation site, tyrosine 416, are not essential for uPA gene induction. However, the reduction of uPA mRNA in pp60v-src-transformed cells by 8-bromo cAMP is dependent on tyrosine 416.

scholarly journals Augmented expression of a myeloid-specific protein tyrosine kinase gene (hck) after macrophage activation.

1988 ◽  
Vol 168 (5) ◽  
pp. 1801-1810 ◽  
Author(s):  
S F Ziegler ◽  
C B Wilson ◽  
R M Perlmutter
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Cell Activity ◽  
Myeloid Cell ◽  
Specific Protein ◽  
Protein Tyrosine Kinases ◽  
Kinase Gene ◽  
Protein Tyrosine ◽  
Family Protein

Protein tyrosine kinases are thought to participate in signal transduction pathways in a variety of cell types. Recent studies have identified a new src family protein tyrosine kinase (hck) that is preferentially expressed in myeloid cells. To examine the hypothesis that this kinase may regulate myeloid cell activity, antisera were generated that define the 59-kD product of the hck gene. Functional activation of human cultured macrophages with LPS augmented the expression of hck transcripts and of p59hck, but decreased the level of transcripts encoded by the closely related c-fgr protooncogene. Thus these two structurally similar src family kinases almost certainly subserve distinct functions. Reasoning from the known properties of the src family protein tyrosine kinases, it is likely that the products of these two protooncogenes assist in regulating the behavior of activated phagocytes.

cDNA cloning and characterization of eck, an epithelial cell receptor protein-tyrosine kinase in the eph/elk family of protein kinases

1990 ◽  
Vol 10 (12) ◽  
pp. 6316-6324
Author(s):  
R A Lindberg ◽  
T Hunter
Keyword(s):  
Protein Kinase ◽  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Catalytic Domain ◽  
Protein Tyrosine Kinases ◽  
Receptor Protein ◽  
Protein Tyrosine ◽  
Kinase Catalytic Domain ◽  
Receptor Protein Tyrosine Kinase

A human epithelial (HeLa) cDNA library was screened with degenerate oligonucleotides designed to hybridize to highly conserved regions of protein-tyrosine kinases. One cDNA from this screen was shown to contain a putative protein-tyrosine kinase catalytic domain and subsequently used to isolate another cDNA from a human keratinocyte library that encompasses the entire coding region of a 976-amino-acid polypeptide. The predicted protein has an external domain of 534 amino acids with a presumptive N-terminal signal peptide, a transmembrane domain, and a cytoplasmic domain of 418 amino acids that includes a canonical protein-tyrosine kinase catalytic domain. Molecular phylogeny indicates that this protein kinase is closely related to eph and elk and that this receptor family is more closely related to the non-receptor protein-tyrosine kinase families than to other receptor protein-tyrosine kinases. Antibodies raised against a TrpE fusion protein immunoprecipitated a 130-kDa protein that became phosphorylated on tyrosine in immune complex kinase assays, indicating that this protein is a bona fide protein-tyrosine kinase. Analysis of RNA from 13 adult rat organs showed that the eck gene is expressed most highly in tissues that contain a high proportion of epithelial cells, e.g., skin, intestine, lung, and ovary. Several cell lines of epithelial origin were found to express the eck protein kinase at the protein and RNA levels. Immunohistochemical analysis of several rat organs also showed staining in epithelial cells. These observations prompted us to name this protein kinase eck, for epithelial cell kinase.

scholarly journals The FER gene is evolutionarily conserved and encodes a widely expressed member of the FPS/FES protein-tyrosine kinase family.

1989 ◽  
Vol 9 (12) ◽  
pp. 5722-5725 ◽  
Author(s):  
T Pawson ◽  
K Letwin ◽  
T Lee ◽  
Q L Hao ◽  
N Heisterkamp ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Gene Product ◽  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Protein Tyrosine Kinases ◽  
Protein Tyrosine ◽  
New Family ◽  
Evolutionarily Conserved ◽  
Conserved Gene ◽  
Nonreceptor Protein Tyrosine Kinases

We have recently isolated human and rat cDNAs (designated FER and flk, respectively) which encode nonreceptor protein-tyrosine kinases which are very similar to one another and related in sequence and domain structure to the c-fps/fes gene product. We show that FER and flk are human and rat counterparts of an evolutionarily conserved gene, hereafter termed FER regardless of species. The human and rat FER genes encode a widely expressed 94-kilodalton protein-tyrosine kinase which is antigenically related to the fps/fes protein-tyrosine kinase. The structural and antigenic similarities between the FER and fps/fes proteins suggest that they are members of a new family of nonreceptor protein-tyrosine kinases.

Involvement of Protein Tyrosine Kinases and Phosphatases in Uptake and Intracellular Replication of Virulent and Avirulent Leishmania donovani Promastigotes in Mouse Macrophage Cells

2002 ◽  
Vol 22 (3-4) ◽  
pp. 395-406 ◽  
Author(s):  
Debjani Ghosh ◽  
Prasanta Chakraborty
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Actin Polymerization ◽  
Protein Tyrosine Kinase ◽  
Leishmania Donovani ◽  
Protein Tyrosine Kinases ◽  
Dependent Manner ◽  
Intracellular Replication ◽  
Protein Tyrosine ◽  
Macrophage Cells

In this study we show that protein tyrosine kinases (PTKs) and also protein tyrosine phosphatases are involved in the uptake of virulent and avirulent Leishmania donovani promastigotes by macrophage cells. Protein tyrosine kinase inhibitors such as genistein or tyrphostin 25 decrease parasite uptake in a dose-dependent manner. Addition of sodium orthovanadate, a protein tyrosine phosphatase inhibitor, prior to infection significantly increases parasite internalization. A similar uptake profile was observed with both virulent and avirulent L. donovani promastigotes. Treatment of macrophages with cytochalasin B, an inhibitor of actin polymerization prevents promastigote uptake, indicating that a tyrosine kinase induced actin polymerization signal may be necessary for the entry of the parasites. In contrast, neither genistein nor tyrphostin significantly reduce intracellular replication of this pathogen or nitric oxide production, suggesting that the PTK-mediated signal is not related to the ultimate virulence mechanism associated with intracellular replication of this pathogen. These data collectively suggest that protein tyrosine kinase mediated entry of L. donovani promastigotes into macrophages is not a virulence-associated event.

Intraspecific and interspecific interactions among proteins regulating exopolysaccharide synthesis in Streptococcus thermophilus, Streptococcus iniae, and Lactococcus lactis subsp. cremoris and the assessment of potential lateral gene transfer

2011 ◽  
Vol 57 (12) ◽  
pp. 1002-1015 ◽  
Author(s):  
Angela D. Cefalo ◽  
Jeffery R. Broadbent ◽  
Dennis L. Welker
Keyword(s):  
Tyrosine Kinase ◽  
Protein Interactions ◽  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Interspecific Interactions ◽  
Streptococcus Thermophilus ◽  
The Other ◽  
Protein Tyrosine Kinases ◽  
Streptococcus Iniae ◽  
Protein Tyrosine

Using the yeast two-hybrid system, intraspecific protein interactions were detected in Streptococcus iniae and Lactococcus lactis subsp. cremoris between the transmembrane activation protein (CpsC and EpsA, respectively) and the protein tyrosine kinase (CpsD and EpsB, respectively), between two protein tyrosine kinases, and between the protein tyrosine kinase and the phosphotyrosine phosphatase (CpsB and EpsC, respectively). For each of these intraspecific interactions, interspecific interactions were also detected when one protein was from S. iniae and the other was from Streptococcus thermophilus . Interactions were also observed between two protein tyrosine kinases when one protein was from either of the Streptococcus species and the other from L. lactis subsp. cremoris. The results and sequence comparisons performed in this study support the conclusion that interactions among the components of the tyrosine kinase – phosphatase regulatory system are conserved in the order Lactobacillales and that interspecific genetic exchanges of the genes that encode these proteins have the potential to form functional recombinants. A better understanding of intraspecific and interspecific protein interactions involved in regulating exopolysaccharide biosynthesis may facilitate construction of improved strains for industrial uses as well as identification of factors needed to form functional regulatory complexes in naturally occurring recombinants.

scholarly journals Myristoylation and differential palmitoylation of the HCK protein-tyrosine kinases govern their attachment to membranes and association with caveolae.

1995 ◽  
Vol 15 (7) ◽  
pp. 3507-3515 ◽  
Author(s):  
S M Robbins ◽  
N A Quintrell ◽  
J M Bishop
Keyword(s):  
Fatty Acids ◽  
Plasma Membrane ◽  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Protein Tyrosine Kinases ◽  
Protein Tyrosine ◽  
Lipid Modifications ◽  
Molecular Weights ◽  
Protein Palmitoylation

The human proto-oncogene HCK encodes two versions of a protein-tyrosine kinase, with molecular weights of 59,000 (p59hck) and 61,000 (p61hck). The two proteins arise from a single mRNA by alternative initiations of translation. In this study, we explored the functions of these proteins by determining their locations within cells and by characterizing lipid modifications required for the proteins to reach those locations. We found that p59hck is entirely associated with cellular membranes, including the organelles known as caveolae; in contrast, only a portion of p61hck is situated on membranes, and none is detectable in preparations of caveolae. These distinctions can be attributed to differential modification of the two HCK proteins with fatty acids. Both proteins are at least in part myristoylated, p59hck more so than p61hck. In addition, however, p59hck is palmitoylated on cysteine 3 in the protein. Palmitoylation of the protein requires prior myristoylation and, in turn, is required for targeting to caveolae. These findings are in accord with recent reports for other members of the SRC family of protein-tyrosine kinases. Taken together, the results suggest that HCK and several of its relatives may participate in the functions of caveolae, which apparently include the transduction of signals across the plasma membrane to the interior of the cell.

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