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.

raplB 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, raplB is membrane-associated in resting platelets, presumably through polyisoprenylation.
The studies presented here were undertaken to determine the subcellular changes in raplB 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, raplB was present in the 100,000 x g supernatant fraction.
In contrast, in platelets activated with 1 unit/ ml a-thrombin or with the calcium ionophore, A23187, raplB was quantitatively recovered in the 10,000 x g cytoskeleton fraction. raplB 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 raplB associates with the cytoskeleton during cell activation.
Low molecular weight GTP-binding proteins are ubiquitous cellular components which function as molecular switches early in the signal transduction pathways for cell growth and differentiation (l-3 protein through a nucleotide exchange reaction. Conversion back to the inactive GDP-ligated protein is facilitated by cytosolic proteins known as GTPase-activating proteins, which enhance the endogenous GTPase activity of the low molecular weight proteins (4). To date, more than 20 distinct low molecular weight G proteins' have been identified (5). The prototype for these proteins is p21", the product of the ras protooncogene.
The subcellular localization of the low molecular weight G proteins appears to be essential for their activity. Two sites of membrane localization have been identified. ~21'" and its homologs are localized to the cytoplasmic face of the plasma membrane, and this localization is absolutely required for transforming activity (6). Localization to the plasma membrane is a complex process that involves a series of posttranslational modifications resulting in covalent attachment of a polyisoprenyl group, typically farnesyl, to the C-terminal cysteine residue (7). The signal for polyisoprenylation is the Cterminal propeptide sequence, Cys-Ali-Ali-Xaa (where Ali is any aliphatic amino acid and Xaa is any amino acid). Following polyisoprenylation, the three C-terminal amino acids (Ali-Ali-Xaa) are proteolytically removed and the C-terminal cysteine is carboxymethylated to yield the mature protein with a polyisoprenylated C-terminal cysteine residue. Mutations of the C-terminal cysteine residue block both polyisoprenylation and membrane localization, as well as the transforming activity (6,8). The localization of these proteins to the plasma membrane is presumed to be essential for their signal-transducing activity. Another group of low molecular weight G proteins, represented by the yeast proteins Sec4 and YPTl, have been localized to internal membranes (9,10). These proteins lack the Cys-Ali-Ali-Xaa signal required for polyisoprenylation but have a C-terminal cysteine residue. It is not known if these proteins are polyisoprenylated or palmitylated.
Recently, a 22-kDa substrate for CAMP-dependent protein kinase in human blood platelets, previously termed p22 or thrombolamban (ll-16), was identified as a low molecular weight G protein. This identification was based on evidence that the protein hydrolyzed GTP and was recognized by antibodies against the GTP-binding domain of ~21" (17-21). Partial protein sequence for the platelet protein (22-24)' was identical with raplB (26), a member of the rus superfamily also known as smg-p21B (17) or m22KG(I) (18). Like p21", raplB in platelets is membrane-associated (19, 21) and has a consensus polyisoprenylation sequence (23-24).* RaplB is phosphorylated in intact platelets by prostaglandin I2 or lipid soluble analogs of CAMP and in membrane fractions by type I CAMP-dependent protein kinase or the catalytic subunit of CAMP-dependent protein kinase (ll-16,20). The phosphorylated amino acid was shown to be a serine (16), and the sequence at the phosphorylation site has been identified as -Arg-Lys-Lys-Ser-Ser-Cys-COOH (24).' Phosphorylation of raplB in intact platelets results in translocation of the protein from a membrane fraction to a cytosolic fraction (19 rap1 B Associates with the Platelet Cytoskeleton phorylation had no effect on the GTPase activity of raplB, and the phosphorylation of GTP-and GDP-bound forms of raplB was kinetically similar (17). Because of its potential importance in cell activation and its known membrane localization, the present study was undertaken to examine the cellular localization of raplB in platelets during activation and was based on several observations. First, since raplB is membrane-associated, probably through either palmitylation or polyisoprenylation of the Cterminal cysteine residue, it was important to determine what happens to the protein during normal cell function. Second, the site of phosphorylation of raplB is remarkably close to the site of membrane attachment (24)," suggesting that membrane attachment may be a regulatable event. Third, several laboratories have reported that palmitylated proteins, including unidentified proteins in the 18-24-kDa range, are incorporated into the platelet cytoskeleton when platelets are activated (27). Finally, recent evidence suggests that some members of the ra.s family of proteins can be associated with intracellular membrane fractions, such as the Golgi apparatus and secretory vesicles (9,10). The results obtained here demonstrate that raplB associates with the cell cytoskeleton during platelet activation and suggest a possible role for this cytoskeleton interaction in platelet function.
Platelets were prepared by differential centrifugation from fresh human blood as described previously (30)

RESULTS
To determine if raplB was a component of the activationdependent cytoskeleton, resting platelets and platelets activated by the addition of 1 unit/ml cu-thrombin were lysed in "lysis buffer" and then fractionated by differential centrifugation to recover a 10,000 x g pellet containing the activationdependent cytoskeleton, a 100,000 X g pellet containing the membrane skeleton, and a 100,000 x g supernatant containing solubilized membrane components and cytosolic proteins (31,32). The results are presented in Fig. 1. In control platelets (panel A), raplB was present in the 100,000 X g supernatant (lanes 7 and 8) and was absent from the cytoskeleton (lanes 3 and 4) and the membrane skeleton (lanes 5 and 6). As previously reported, the activation-dependent cytoskeleton is not fully assembled in resting platelets and contains mostly actin (31). Upon activation rctplB Associates with the Platelet Cytoskeleton   19407 to the known cytoskeletal components actin-binding protein, myosin, cu-actinin, and actin, respectively, were observed in the cytoskeletal fraction (lane 3). Protein bands were also seen at 55-60 kDa, corresponding to the (Y, 6, and y chains of fibrinogen, and at 22 kDa. On Western blots, raplB was found entirely in the cytoskeletal fraction (lone 4) and had the same electrophoretic mobility as the 22-kDa band seen in Coomassie-stained lanes. Fractions containing the membrane skeleton (panel B lanes 5 and 6) or the supernatant (lanes 7 and 8) did not contain detectable raplB by either Western blot with M90 or by protein stain. When platelets were activated with the calcium ionophore A23187, results similar to those depicted in Fig. 1 were obtained (data not shown).
The function of raplB and other low molecular weight Gproteins in platelets remains unclear. Our demonstration that raplB incorporates into the cytoskeleton during cell activation suggests that it functions less in initial signal transduction and more in subsequent events. Indeed, if the proposed roles of Sec4 and YPTl in yeast (9,10) can be extended to other systems, one might speculate that raplB acts as a targeting mechanism in the cytoskeleton, perhaps mediating protein-protein interactions or the interaction of the cytoskeleton with another cellular component, such as secretory DISCUSSION We present evidence that raplB, a ras-like protein that both binds GTP and is a substrate for CAMP-dependent protein kinase, is incorporated into the cytoskeleton of human platelets during cell activation. These data provide direct evidence for a possible role of raplB in cytoskeletal assembly and amplify previous studies in other cells indirectly implicating low molecular weight G proteins in cytoskeletal function (35, 36).
The cellular skeleton of platelets contains several structural components (31,32,37). The membrane skeleton consists of short actin filaments cross-linked by actin-binding protein and is linked to the glycoprotein Ib-IX complex in the plasma membrane. It provides the platelet with its shape and plays an important role in determining the organization and function of the GPIb-IX complex in the plasma membrane. This structure is present in resting platelets but is disrupted following platelet activation, at least in part due to the cleavage of actin-binding protein by the calcium-activated protease (38, 39), calpain, resulting in changes in platelet shape and increased mobility of membrane proteins. The cytoplasmic skeleton or cytoskeleton from resting platelets consists primarily of a mesh of actin filaments. When platelets are activated, actin, myosin, and actin-binding protein are incorporated into the cytoskeleton. Our results indicate that raplB is a component of the activation-dependent cytoskeleton, but not of the membrane skeleton, even though raplB is membranebound in resting platelets. Furthermore, in contrast to other cytoskeletal proteins such as actin-binding protein and actin, which are only partly incorporated into the cytoskeleton, all of the immunodetectable raplB becomes incorporated into the cytoskeleton during activation. This quantitative incorporation of raplB is distinct and suggests that the cytoskeletal interaction is an important one.
In addition to raplB, two other cytoskeletal proteins have been identified as CAMP-dependent protein kinase substrates: the /3 subunit of GPIb (40), which is present in the membrane skeleton, and actin-binding protein (41). Cyclic AMP-dependent phosphorylation of GPIbfi in intact platelets did not inhibit agonist-stimulated aggregation, secretion, or phosphorylation of myosin light chain or ~47. However, agoniststimulated actin polymerization was inhibited suggesting that phosphorylation of GPBIbp influenced cytoskeletal assembly (25). In preliminary studies, we have tried to examine the effect of phosphorylation on raplB incorporation into the cytoskeleton, but treatment with agents that increased CAMP levels inhibited cytoskeletal assembly.3 The identification of three proteins involved in the CAMP pathway as components of the platelet cytoskeleton is remarkable and suggests that at least one of the inhibitory mechanisms of CAMP is through an effect on the cytoskeleton. granules.
In summary, the membrane interactions of raplB in platelets appear to be complex but may ultimately yield important clues to the function of the low molecular G proteins. In resting platelets, raplB is associated with the cell membrane, probably the plasma membrane, and probably through a polyisoprenyl group. Whether or not this involves changes in the posttranslational modification of raplB remains to be determined. When the cell is inhibited by CAMP, raplB is translocated from the membrane to a cytosolic location (19). When the cell is activated, raplB becomes incorporated into the cytoskeleton. This bidirectional translocation may be essential for normal cell function.