Use of an activation-specific probe to show that Rap1A and Rap1B display different sensitivities to activation by forskolin in rat1 cells.
Rap1A and Rap1B are small GTPases of the Ras superfamily whose activation can be measured using a probe that interacts specifically with the GTP-bound forms of Rap1A and Rap1B. Using this procedure we demonstrate that the cyclic AMP-elevating agent forskolin activates both Rap1A and Rap1B in Rat1 cells. Whilst the protein kinase A inhibitor H89 ablated the ability of forskolin to cause cAMP response element binding protein (CREB) phosphorylation in Rat1 cells, it did not affect the ability of forskolin to activate either Rap1A and Rap1B. Forskolin differentially activated Rap1A and Rap1B isoforms in a time- and dose-dependent manner. The cAMP-specific type 4 family phosphodiesterase inhibitor rolipram potentiated the rate of activation of both Rap1A and Rap1B by forskolin challenge of Rat1 cells. Challenge of Rat1 cells with rolipram alone was able to elicit the phosphorylation of CREB but not activation of either Rap1A or Rap1B.
McPhee
I
I
Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Wolfson and Davidson Buildings, IBLS, University of Glasgow, G12 8QQ, Glasgow, UK.
Houslay
M D
MD
Yarwood
S J
SJ
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology*; D017931:DNA Primers; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D051381:Rats; D020889:Rolipram / Q000494:pharmacology; D020668:rap GTP-Binding Proteins / Q000378:metabolism*
D017931:DNA Primers; D020690:GTPase-Activating Proteins; C116071:rap1a-GTPase activating proteins, human; C485482:smg p21 GAP1 protein, human; C485483:smg p21 GAP2 protein, human; D005576:Colforsin; D015124:8-Bromo Cyclic Adenosine Monophosphate; C492135:Rap1b protein, rat; D020668:rap GTP-Binding Proteins; D020889:Rolipram