Beta2-chimaerin is a high affinity receptor for the phorbol ester tumor promoters.
Beta2-chimaerin, a member of the GTPase-activating proteins for the small GTP-binding protein p21Rac, possesses a single cysteine-rich domain with high homology to those implicated in phorbol ester and diacylglycerol binding in protein kinase C (PKC) isozymes. We have expressed beta2-chimaerin in Sf9 insect cells using the baculovirus expression system and determined that, like PKCs, beta2-chimaerin binds phorbol esters with high affinity in the presence of phosphatidylserine as a cofactor. Scatchard plot analysis using the radioligand [3H]phorbol 12,13-dibutyrate revealed a dissociation constant of 1.9 +/- 0.2 nM for beta2-chimaerin. Likewise, beta2-chimaerin is a high affinity receptor for the bryostatins, a class of atypical PKC activators. A detailed comparison of structure-activity relations using several phorbol ester analogs revealed striking differences in binding recognition between beta2-chimaerin and PKCalpha. Although the diacylglycerol 1-oleoyl-2-acetylglycerol binds with similar potency to both beta2-chimaerin and PKCalpha, the mezerein analog thymeleatoxin has 56-fold less affinity for binding to beta2-chimaerin. To establish whether beta2-chimaerin responds to phorbol esters in cellular systems, we overexpressed beta2-chimaerin in COS-7 cells and monitored its subcellular distribution after phorbol ester treatment. Interestingly, as described previously for PKC isozymes, beta2-chimaerin translocates from cytosolic to particulate fractions as a consequence of phorbol ester treatment. Our results demonstrate that beta2-chimaerin is a novel target for the phorbol ester tumor promoters. The expansion of the family of phorbol ester receptors strongly suggests a potential for the "non-kinase" receptors as cellular mediators of the phorbol ester responses.
Caloca
M J
MJ
Center for Experimental Therapeutics and Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6100, USA.
Fernandez
N
N
Lewin
N E
NE
Ching
D
D
Modali
R
R
Blumberg
P M
PM
Kazanietz
M G
MG
D000818:Animals; D001692:Biological Transport; D019556:COS Cells; D002273:Carcinogens / Q000378:metabolism*; D002451:Cell Compartmentation; D002460:Cell Line; D004789:Enzyme Activation; D009363:Neoplasm Proteins / Q000378:metabolism*; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism*; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D018411:Spodoptera
D002273:Carcinogens; D009363:Neoplasm Proteins; D011994:Recombinant Proteins; C079844:beta-chimaerin; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C