Cell cycle-specific induction of an 89 kDa serine/threonine protein kinase activity in Trypanosoma brucei.
The cell cycle compartmentalization of specific activities of the protozoan parasite Trypanosoma brucei has remained unexplored due to the lack of a cell synchronization protocol. We report here that stationary phase cells stimulated to enter the cell cycle showed significant synchrony through the first cycle. The pattern of tyrosine phosphorylated proteins, known to undergo alterations during trypanosome development, showed only moderate changes as quiescent cells entered the cycle, particularly an increase in a 77 kDa species. However, the activity of an 89 kDa protein kinase (SPK89), previously demonstrated to be restricted to the proliferative stages of the parasite's life cycle, markedly increased as the population entered S phase. Cell sorting experiments demonstrated that SPK89 activity was highest in S phase cells and moderate in G2/M cells. The entry into S phase and increased SPK89 activity did not depend on serum factors but required protein synthesis for a discrete period after stimulation. Various modulators of protein phosphorylation were tested to determine their effects on progression to S and SPK89 activity. Only staurosporine and genistein were effective. However, both of these compounds inhibited virtually all protein phosphorylation and protein synthesis in the parasites. Thus these drugs cannot be used as specific protein kinase inhibitors in trypanosomes.
Gale
M
M
Seattle Biomedical Research Institute, WA 98105.
Carter
V
V
Parsons
M
M
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D002396:Catechols / Q000494:pharmacology; D002453:Cell Cycle / Q000502:physiology*; D002455:Cell Division / Q000187:drug effects; D003513:Cycloheximide / Q000494:pharmacology; D004247:DNA / Q000378:metabolism; D004790:Enzyme Induction; D004988:Ethers, Cyclic / Q000494:pharmacology; D005434:Flow Cytometry; D019833:Genistein; D007529:Isoflavones / Q000494:pharmacology; D007546:Isoquinolines / Q000494:pharmacology; D008970:Molecular Weight; D009570:Nitriles / Q000494:pharmacology; D019319:Okadaic Acid; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine; D010879:Piperazines / Q000494:pharmacology; D011489:Protein Denaturation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D016196:S Phase / Q000187:drug effects; D019311:Staurosporine; D014346:Trypanosoma brucei brucei / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis; D020032:Tyrphostins
D000470:Alkaloids; D002396:Catechols; D004988:Ethers, Cyclic; D007529:Isoflavones; D007546:Isoquinolines; D009570:Nitriles; D010750:Phosphoproteins; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D020032:Tyrphostins; C060152:tyrphostin 47; D019319:Okadaic Acid; D019000:Phosphotyrosine; D014443:Tyrosine; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; C053177:N-(2-(methylamino)ethyl)-5-isoquinolinesulfonamide; D004247:DNA; D003513:Cycloheximide; D019833:Genistein; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C; D019311:Staurosporine