Abstract 1271: Aberrant activation of Spleen Tyrosine Kinase in ovarian cancer identified through a global phosphorylation profiling of protein tyrosine kinases

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.

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Kinome Array Profiling of Patient-Derived Pancreatic Ductal Adenocarcinoma Identifies Differentially Active Protein Tyrosine Kinases

International Journal of Molecular Sciences ◽

10.3390/ijms21228679 ◽

2020 ◽

Vol 21 (22) ◽

pp. 8679 ◽

Cited By ~ 1

Author(s):

Justin F. Creeden ◽

Khaled Alganem ◽

Ali S. Imami ◽

F. Charles Brunicardi ◽

Shi-He Liu ◽

...

Keyword(s):

Pancreatic Cancer ◽

Growth Factor ◽

Tyrosine Kinase ◽

Pancreatic Ductal Adenocarcinoma ◽

Tyrosine Kinases ◽

Growth Factor Receptor ◽

Protein Tyrosine Kinases ◽

Ductal Adenocarcinoma ◽

Active Protein ◽

Protein Tyrosine

Pancreatic cancer remains one of the most difficult malignancies to treat. Minimal improvements in patient outcomes and persistently abysmal patient survival rates underscore the great need for new treatment strategies. Currently, there is intense interest in therapeutic strategies that target tyrosine protein kinases. Here, we employed kinome arrays and bioinformatic pipelines capable of identifying differentially active protein tyrosine kinases in different patient-derived pancreatic ductal adenocarcinoma (PDAC) cell lines and wild-type pancreatic tissue to investigate the unique kinomic networks of PDAC samples and posit novel target kinases for pancreatic cancer therapy. Consistent with previously described reports, the resultant peptide-based kinome array profiles identified increased protein tyrosine kinase activity in pancreatic cancer for the following kinases: epidermal growth factor receptor (EGFR), fms related receptor tyrosine kinase 4/vascular endothelial growth factor receptor 3 (FLT4/VEGFR-3), insulin receptor (INSR), ephrin receptor A2 (EPHA2), platelet derived growth factor receptor alpha (PDGFRA), SRC proto-oncogene kinase (SRC), and tyrosine kinase non receptor 2 (TNK2). Furthermore, this study identified increased activity for protein tyrosine kinases with limited prior evidence of differential activity in pancreatic cancer. These protein tyrosine kinases include B lymphoid kinase (BLK), Fyn-related kinase (FRK), Lck/Yes-related novel kinase (LYN), FYN proto-oncogene kinase (FYN), lymphocyte cell-specific kinase (LCK), tec protein kinase (TEC), hemopoietic cell kinase (HCK), ABL proto-oncogene 2 kinase (ABL2), discoidin domain receptor 1 kinase (DDR1), and ephrin receptor A8 kinase (EPHA8). Together, these results support the utility of peptide array kinomic analyses in the generation of potential candidate kinases for future pancreatic cancer therapeutic development.

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

Molecular and Cellular Biology ◽

10.1128/mcb.10.12.6244-6256.1990 ◽

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.

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Cytosolic Protein-Tyrosine Kinase Activities in Various Rat Tissues

Annals of Clinical Biochemistry International Journal of Laboratory Medicine ◽

10.1177/000456328902600213 ◽

1989 ◽

Vol 26 (2) ◽

pp. 164-168 ◽

Cited By ~ 14

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.

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Altered Association of Protein Tyrosine Kinases with Postsynaptic Densities after Transient Cerebral Ischemia in the Rat Brain

Journal of Cerebral Blood Flow & Metabolism ◽

10.1097/00004647-200003000-00009 ◽

2000 ◽

Vol 20 (3) ◽

pp. 505-512 ◽

Cited By ~ 36

Author(s):

Herman H. Cheung ◽

Norio Takagi ◽

Lucy Teves ◽

Richard Logan ◽

M. Christopher Wallace ◽

...

Keyword(s):

Cerebral Ischemia ◽

Nmda Receptor ◽

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

Tyrosine Kinases ◽

Transient Cerebral Ischemia ◽

Protein Tyrosine Kinases ◽

Vessel Occlusion ◽

Protein Tyrosine ◽

Tyrosine Kinase 2

Transient cerebral ischemia results in an increase in the tyrosine phosphorylation of proteins associated with postsynaptic densities (PSDs). The authors investigated the possible mechanisms behind this increase by analyzing isolated PSDs for protein tyrosine kinase activity and for the presence of specific tyrosine kinases. Transient (15 minutes) global ischemia was produced in adult rats by four-vessel occlusion, and PSDs were isolated immediately after ischemia or after 20 minutes or 6 hours of reperfusion. Tyrosine phosphorylation of several PSD proteins, including the N-methyl-d-aspartate (NMDA) receptor subunits NR2A and NR2B, was enhanced relative to shams after 20 minutes of reperfusion and underwent a further increase between 20 minutes and 6 hours. The ability of intrinsic PSD tyrosine kinase to phosphorylate PSD proteins, including the NMDA receptor, increased threefold after ischemia. Whereas PSD-associated proline-rich tyrosine kinase 2 (PYK2) and gp145TrkB were elevated immediately after the ischemic event, increases in Src and Fyn were not apparent until 6 hours of reperfusion. The level of PSD-associated pp125FAK decreased after ischemia. The results demonstrate that ischemia results in selective changes in the association of protein tyrosine kinases with the PSD which may account for ischemia-induced increases in the tyrosine phosphorylation of PSD proteins.

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

Molecular and Cellular Biology ◽

10.1128/mcb.13.9.5888 ◽

1993 ◽

Vol 13 (9) ◽

pp. 5888-5897 ◽

Cited By ~ 18

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.

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

Journal of Experimental Medicine ◽

10.1084/jem.168.5.1801 ◽

1988 ◽

Vol 168 (5) ◽

pp. 1801-1810 ◽

Cited By ~ 59

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.

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Src family protein tyrosine kinases induce autoactivation of Bruton's tyrosine kinase.

Molecular and Cellular Biology ◽

10.1128/mcb.15.10.5304 ◽

1995 ◽

Vol 15 (10) ◽

pp. 5304-5311 ◽

Cited By ~ 98

Author(s):

S Mahajan ◽

J Fargnoli ◽

A L Burkhardt ◽

S A Kut ◽

S J Saouaf ◽

...

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Kinases ◽

Enzyme Activation ◽

Protein Tyrosine Kinases ◽

Activation Process ◽

Bruton’S Tyrosine Kinase ◽

Src Kinases ◽

Regulatory Relationship ◽

Bruton's Tyrosine Kinase ◽

Protein Tyrosine

Bruton's tyrosine kinase (Btk) is tyrosine phosphorylated and enzymatically activated following ligation of the B-cell antigen receptor. These events are temporally regulated, and Btk activation follows that of various members of the Src family of protein tyrosine kinases, thus raising the possibility that Src kinases participate in the Btk activation process. We have evaluated the mechanism underlying Btk enzyme activation and have explored the potential regulatory relationship between Btk and Src protein kinases. We demonstrate in COS transient-expression assays that Btk can be activated through intramolecular autophosphorylation at tyrosine 551 and that Btk autophosphorylation is required for Btk catalytic functions. Coexpression of Btk with members of the Src family of protein tyrosine kinases, but not Syk, led to Btk tyrosine phosphorylation and activation. Using a series of point mutations in Blk (a representative Src protein kinase) and Btk, we show that Src kinases activate Btk through an indirect mechanism that requires membrane association of the Src enzymes as well as functional Btk SH3 and SH2 domains. Our results are compatible with the idea that Src protein tyrosine kinases contribute to Btk activation by indirectly stimulating Btk intramolecular autophosphorylation.

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

Molecular and Cellular Biology ◽

10.1128/mcb.10.12.6316-6324.1990 ◽

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.

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