A cytosolic activator of DNA replication is tyrosine phosphorylated in its active form.
Cytosolic extracts from actively dividing lymphoid cells have been shown to induce DNA synthesis in isolated, quiescent nuclei. An initiating factor in such extracts (activator of DNA replication; ADR) is a > 90-kDa aprotinin-binding protein whose activity is inhibitable not only by aprotinin, but also by several other protease inhibitors as well. Although cytosol from non-proliferating lymphocytes is devoid of ADR activity, we have shown that these preparations can be induced to express ADR activity by brief exposure to a membrane-enriched fraction of spontaneously proliferating MOLT-4 cells via a kinase-dependent mechanism. In the present study, we examine the role of tyrosine kinases in this process. Three inhibitors of tyrosine kinases (genistein, kaempferol, and quercetin) can inhibit the in vitro generation of ADR activity. In vitro generation of ADR activity is associated with the de novo phosphorylation of several proteins, many of which are detectable using anti-phosphotyrosine monoclonal antibodies. ADR itself may be tyrosine phosphorylated in active form as immunoprecipitation using such monoclonal antibodies leads to the depletion of its activity. Moreover, immunoprecipitation results in the removal of several de novo tyrosine-phosphorylated proteins, including species at approximately 122, 105, 93, 86, 79, and 65 kDa. A subset of de novo-phosphorylated proteins, migrating at approximately 105, 93, and 70 kDa, also bound to aprotinin, suggesting that at least one of these proteins may represent ADR itself.
Fresa
K L
KL
Department of Pathology, Hahnemann University School of Medicine, Philadelphia, Pennsylvania 19102.
Autieri
M V
MV
Coffman
F D
FD
Georgoff
I
I
Cohen
S
S
D007611:Aprotinin / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004261:DNA Replication; D004305:Dose-Response Relationship, Drug; D005419:Flavonoids; D019833:Genistein; D006801:Humans; D066298:In Vitro Techniques; D007529:Isoflavones / Q000494:pharmacology; D044949:Kaempferols; D010766:Phosphorylation; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D011794:Quercetin / Q000031:analogs & derivatives / Q000494:pharmacology; D015398:Signal Transduction; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D005419:Flavonoids; D007529:Isoflavones; D044949:Kaempferols; D019000:Phosphotyrosine; D014443:Tyrosine; C006552:kaempferol; D007611:Aprotinin; D011794:Quercetin; D019833:Genistein; D011505:Protein-Tyrosine Kinases