Faculty Collaboration Database

Incorporation of Rap 1b into the platelet cytoskeleton is dependent on thrombin activation and extracellular calcium. J Biol Chem 1994 Jun 24;269(25):17257-61

Date

06/24/1994

Pubmed ID

7516336

Scopus ID

2-s2.0-0028301240 (requires institutional sign-in at Scopus site)   33 Citations

Abstract

Rap 1b is a 22-kDa low molecular mass GTP-binding protein which is both a member of the Ras superfamily and a substrate for cAMP-dependent protein kinase. Recently, evidence has been presented to show that Rap 1b is incorporated into the detergent-extracted cytoskeleton of platelets during thrombin-induced activation. The aims of this study were to compare the incorporation of Rap 1b into the detergent-extracted cytoskeleton after activation with different agonists, to examine the role of extracellular calcium on the incorporation of Rap 1b into the cytoskeleton, to investigate the relationship between the association of Rap 1b and other proteins with the cytoskeleton, and to determine the effect of phosphorylation of Rap 1b incorporation into the cytoskeleton. Platelets were activated with thrombin, A23187, phorbol myristate acetate, ADP, epinephrine, and collagen in the presence and absence of calcium. The time dependence of Rap 1b incorporation into the detergent-extracted cytoskeleton was then measured. When platelets were activated by thrombin in the presence of extracellular calcium, conditions which permit aggregation, incorporation of Rap 1b into the detergent-extracted cytoskeleton was biphasic. Approximately 20% of the total cellular Rap 1b incorporated into the cytoskeleton within seconds and was followed by a slower second phase of incorporation. In contrast, when platelets were activated by thrombin in the absence of calcium, conditions which inhibit aggregation, or by the other agents in the presence or absence of calcium, only the initial phase of Rap 1b incorporation into the cytoskeleton was measured. The incorporation of Rap 1b paralleled the incorporation of membrane glycoproteins (GP) IIb/IIIa and PECAM-1, but not the incorporation of pp60c-src. The GTPase-activating protein for Ras (Ras-GAP) did not associate with the detergent-extracted cytoskeleton. Two-dimensional isoelectric focusing SDS-polyacrylamide gel electrophoresis of the total cellular and cytoskeletal Rap 1b showed that unphosphorylated as well as phosphorylated isoforms of Rap 1b were incorporated into the cytoskeleton in the same molar ratio as was present in the intact cell. Furthermore, the rates of incorporation of phosphorylated and unphosphorylated Rap 1b into the cytoskeleton were similar. These experiments show that Rap 1b can regulate events that take place within seconds after activation, such as the initial formation of the cytoskeleton, as well as longer term changes in the cytoskeleton that occur in response to thrombin-induced aggregation. Furthermore, phosphorylation could modulate the (unknown) functions of Rap 1b as a component of the cytoskeleton.

Author List

Fischer TH, Gatling MN, McCormick F, Duffy CM, White GC 2nd

Author

Gilbert C. White MD Professor in the Medicine department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Blood Platelets
Calcium
Cytoskeleton
Electrophoresis, Gel, Two-Dimensional
Enzyme Activation
GTP-Binding Proteins
Humans
In Vitro Techniques
Phosphoproteins
Platelet Aggregation
Proto-Oncogene Proteins pp60(c-src)
Thrombin
rap GTP-Binding Proteins