rap1B, a cAMP-dependent protein kinase substrate, associates with the platelet cytoskeleton.
rap1B is a member of the ras superfamily of low molecular weight GTP binding proteins which constitutes a focal point of GTP and cAMP signal transduction systems. Like other members of this superfamily, rap1B is membrane-associated in resting platelets, presumably through polyisoprenylation. The studies presented here were undertaken to determine the subcellular changes in rap1B localization during cell activation. Activated and unactivated platelets were fractionated by Triton X-100 lysis followed by differential centrifugation to obtain a 10,000 x g cytoskeleton fraction, a 100,000 x g membrane skeleton fraction, and a 100,000 x g supernatant fraction containing solubilized proteins. In unactivated platelets, rap1B was present in the 100,000 x g supernatant fraction. In contrast, in platelets activated with 1 unit/ml alpha-thrombin or with the calcium ionophore, A23187, rap1B was quantitatively recovered in the 10,000 x g cytoskeleton fraction. rap1B was absent from the 100,000 x g fraction containing the membrane skeleton and could not be detected in the 100,000 x g supernatant containing cytosolic proteins and solubilized membrane components. These results indicate that rap1B associates with the cytoskeleton during cell activation.
Fischer
T H
TH
Department of Medicine, Dental Research Center, University of North Carolina, Chapel Hill 27599.
Gatling
M N
MN
Lacal
J C
JC
White
G C
GC
D000595:Amino Acid Sequence; D001792:Blood Platelets / Q000187:drug effects / Q000648:ultrastructure*; D015153:Blotting, Western; D000001:Calcimycin / Q000494:pharmacology; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015539:Platelet Activation / Q000502:physiology*; D011494:Protein Kinases / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology; D020668:rap GTP-Binding Proteins
D000001:Calcimycin; D011494:Protein Kinases; D013917:Thrombin; D019204:GTP-Binding Proteins; D020668:rap GTP-Binding Proteins