Partial purification and characterization of a new p36/40 tyrosine protein kinase from HL-60

Boutin, Jean A.; Ernould, Anne-Pascale; Genton, Annie; Cudennec, Claude A.

doi:10.1016/s0006-291x(89)80131-7

Cited by 10 publications

(5 citation statements)

References 21 publications

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“…These results suggest that the prostatic PTK possesses autophosphorylation sites and is likely to catalyze the phosphorylation of its tyrosyl residues. It is noteworthy that autophosphorylation is a characteristic of almost all described PTKs with few exceptions, such as a p36-40 PTK from HL-60 cells [21] and a p50 CSK from neonatal rat brain [22]. To identify the PTK, enzyme fractions ¢luted from Sephadex G-100 were pooled and further purified by chromatofocusing which, instead of the single peak of activity observed at a pl of 5.5 as reported previously [1], yielded a major peak characterized by a pl of 5.0, and a minor one by a pI of 5.6 (Fig.…”

Section: Results and Discussionmentioning

confidence: 99%

The major form of protein tyrosine kinase in the dog prostate is expressed by a 50 kDa polypeptide

et al. 1992

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We have already reported that the protein tyrosine kinase (PTK) activity in the dog prostate is distributed in cytosolic (75%) and paniculate (Triton X-100.solubilized) fractions and that upon gel filtration, both PTKs migrate as entiti~ of M, 44 009 [(1991) Biochem. Cell. Biol. 69. 146-153]. lterein we demonstrate by immunopre¢ipitation with anti-phosphotyrosine antibodies that the soluble PTK has the ability to undergo self-phosphory, lation. In addition, the polypeptide responsible for that ellzymatic activity has been identified by 2 approaches: (I) a two-dimensional elcetrophoresis, in which the first dimension performed in non-denaturing conditions allowed the localization of the native enzyme, while the second dimension ($DS-PAGE) permitted the analysis of alkali-resistant phosphoproteins corresponding to the activity; (2) protein renaturation after SDS-PAGE followed by in situ phosphorylation (with [)'-'~"P]ATP) of polyGT el~trophoresed together with the enzyme preparation; the exclusive presence of the radiolabeled phosphotyrosine in the renatured protein confirmed its enzymatic nature. Using these methods, the major form of PTK in the dog prostate was shown to he expressed by a 50 kDa polypeptide which possesses autophosphorylation sites and which is present in the cytosol as an active monomer.

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Section: Results and Discussionmentioning

confidence: 99%

The major form of protein tyrosine kinase in the dog prostate is expressed by a 50 kDa polypeptide

et al. 1992

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“…Here we report the purification of a tyrosine-specific protein kinase from the particulate fraction of rat spleen and its identification as the catalytic domain of p72 syk (CDp72 syk ), by using an antibody recognizing the kinase's ' linker ' region [8]. The subsequent determination of the biochemical properties of the kinase allows a comparative study with other similarly sized tyrosine kinases previously reported [1,5,9]. Using a newly developed assay, we demonstrate that the catalytic domain of p72 syk serves as a substrate for protein kinase C (PKC) in itro, with PKC phosphorylation having an activating effect on CDp72 syk .…”

Section: Introductionmentioning

confidence: 99%

Purification of catalytic domain of rat spleen p72syk kinase and its phosphorylation and activation by protein kinase C

Borowski

Heiland

Kornetzky

et al. 1998

Biochemical Journal

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The catalytic domain of p72(syk) kinase (CDp72(syk)) was purified from a 30000 g particulate fraction of rat spleen. The purification procedure employed sequential chromatography on columns of DEAE-Sephacel and Superdex-200, and elution from HA-Ultrogel by chloride. The analysis of the final CDp72(syk) preparation by SDS/PAGE revealed a major silver-stained 40 kDa protein. The kinase was identified by covalent modification of its ATP-binding site with [14C]5'-fluorosulphonylbenzoyladenosine and by immunoblotting with a polyclonal antibody against the 'linker' region of p72(syk). By using poly(Glu4, Tyr1) as a substrate, the specific activity of the enzyme was determined as 18.5 nmol Pi/min per mg. Casein, histones H1 and H2B and myelin basic protein were efficiently phosphorylated by CDp72(syk). The kinase exhibited a limited ability to phosphorylate random polymers containing tyrosine residues. CDp72(syk) autophosphorylation activity was associated with an activation of the kinase towards exogenous substrates. The extent of activation was dependent on the substrates added. CDp72(syk) was phosphorylated by protein kinase C (PKC) on serine and threonine residues. With a newly developed assay method, we demonstrated that the PKC-mediated phosphorylation had a strong activating effect on the tyrosine kinase activity of CDp72(syk). Studies extended to conventional PKC isoforms revealed an isoform-dependent manner (alpha > betaI = betaII > gamma) of CDp72(syk) phosphorylation. The different phosphorylation efficiencies of the PKC isoforms closely correlated with the ability to enhance the tyrosine kinase activity.

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“…Very early on, we became interested also in kinases and some of their modifying enzymes (e.g., NMT, see above). Indeed, we started our program by choosing to explore the main tyrosine protein kinase expressed in the HL60 cancer cell line [50,51] and purification was the only option at a time when cloning was rather rare.…”

Section: Cloning/expressing/purifying the Targetsmentioning

confidence: 99%

On the Organization of a Drug Discovery Platform

Nosjean¹,

Ferry²

2018

Drug Discovery - Concepts to Market

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Some of the most exciting parts of work in the pharmaceutical industry are the steps leading up to drug discovery. This process can be oversimplified by describing it as a screening campaign involving the systematic testing of many compounds in a test relevant to a given pathology. This naïve description takes place without taking into consideration the numerous key steps that led up to the screening or the steps that might follow. The present chapter describes this whole process as it was conducted in our company during our early drug discovery activities. First, the purpose of the procedures is described and rationalized. Next follows a series of mostly published examples from our own work illustrating the various steps of the process from cloning to biophysics, including expression systems and membrane-bound protein purifications. We believe that what is described here presents an example of how pharmaceutical industry research can organize its platform(s) when the goal is to find and qualify a new preclinical drug candidate using cutting-edge technologies and a lot of hard work.

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Partial purification and characterization of a new p36/40 tyrosine protein kinase from HL-60

Cited by 10 publications

References 21 publications

The major form of protein tyrosine kinase in the dog prostate is expressed by a 50 kDa polypeptide

The major form of protein tyrosine kinase in the dog prostate is expressed by a 50 kDa polypeptide

Purification of catalytic domain of rat spleen p72syk kinase and its phosphorylation and activation by protein kinase C

On the Organization of a Drug Discovery Platform

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