WAVE/Scars in platelets

Abstract

Using specific antibodies against isoforms of WAVE (WASP [Wiskott-Aldrich syndrome protein] family Verprolinhomologous protein, also called Scar), we demonstrated that human platelets express all 3 isoforms. With the use of an in vitro pull-down technique, the src homology 3 (SH3) domain of insulin receptor substrate p53 (IRSp53) precipitated WAVE2 from platelet lysates more efficiently than did profilin I. The opposite was true for WAVE1, and neither precipitated WAVE3, suggesting that WAVE isoforms have different affinities to these ligands, while the SH3 domain of abl binds to all 3 isoforms. The 3 WAVE isoforms were distributed in the actinrich Triton X-100-insoluble pellets following platelet aggregation induced by thrombin receptor-activating peptide. We also found that all 3 WAVE isoforms are substrates for calpain in vivo and in vitro. Although portions of these 3 isoforms were commonly distributed in the actinand actin-related protein 2 and 3 (Arp2/3)-rich edge of the lamellipodia in spreading platelets, only WAVE2 remained in the cell fringe following detergent extraction or fixation of the cells. Finally, by mass spectrometry, we found that the proteins, which reportedly interact with WAVE/ Scars, are present in platelets. These data suggest that the 3 WAVE isoforms exhibit common and distinct features and may potentially be involved in the regulation of actin cytoskeleton in platelets. IntroductionReorganization of cortical actin filaments plays a critical role in cell movement and pattern formation. 1,2 WASP (Wiskott-Aldrich syndrome protein), N-WASP, and WAVE (WASP family Verprolinhomologous protein, or Scar), called WASP family proteins, are likely to regulate cortical actin filament reorganization in response to extracellular stimuli, although not all of these proteins have been linked to actin polymerization. 1,2 Each of these proteins has a verprolin-homology (V) domain, cofilin-homology (C) domain, and an acidic (A) region at the C-terminus, which are necessary to enhance the intrinsic actin polymerization activity of the actinrelated protein 2 and 3 (Arp2/3) complex. [1][2][3][4][5][6][7][8][9] Platelets are essential for normal hemostasis and have an unusually high actin content (0.5 mM). 10,11 In suspension, platelets are rapidly transformed from disks to spheres with spiny protrusions upon stimulation by agonists such as adenosine diphosphate, collagen, and thrombin. [10][11][12][13][14][15] This "shape change" is inhibited by cytochalasins, which prevent actin polymerization. [10][11][12][13][14][15] Platelets also adhere onto various surfaces and rapidly spread, a process that is also inhibited by cytochalasins. Thus, actin polymerization is pivotal in the rapid morphologic transformation of platelets in the initial phase of hemostasis. Among the WASP and WAVE family proteins, platelets express WASP, which becomes strongly tyrosine phosphorylated upon stimulation by collagen or following crosslinking of CD32, a low-affinity Fc receptor for immunoglobulin G (IgG). 16 However, despite the...

The Reversion-inducing Cysteine-rich Protein with Kazal Motifs (RECK) Interacts with Membrane Type 1 Matrix Metalloproteinase and CD13/Aminopeptidase N and Modulates Their Endocytic Pathways

“…Next, we addressed whether RECK can influence the aminopeptidase activity of CD13. Introduction of exogenous RECK reduced the cell surface aminopeptidase activity to a level comparable with that in cells that were treated with a non-cytotoxic concentration of bestatin, a specific aminopeptidase inhibitor (31) (Fig. 3E).…”

“…Mass Spectrometry-Mass spectrometric identification of proteins was performed by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry as described previously (31).…”

Syk Regulates Multiple Signaling Pathways Leading to CX3CL1 Chemotaxis in Macrophages

“…Similar to the results shown here for CX3CL1, WAVE2 and WASP are known downstream effectors of Rac and Cdc42 that are required for actin remodeling and chemotaxis in macrophages in response to CSF-1 (20,24,30,32). WAVE2 does not bind directly to Rac and IRSp53 was reported to be an important candidate linking WAVE2 to Rac1 (61,62). Consistent with IRSp53 in mediating the interaction between Rac and WAVE2, WAVE2 was present in a complex with Rac1 and IRSp53 in response to CX3CL1 and disruption of this interaction using a dominant-negative form of IRSp53-inhibited membrane protrusions in response CX3CL1.…”

Membrane targeting of WAVE2 is not sufficient for WAVE2-dependent actin polymerization: a role for IRSp53 in mediating the interaction between Rac and WAVE2

“…Although WAVE2 has a well-documented function in cell migration, regulation of WAVE2 has proven to be very complicated as Rac1 does not bind directly to WAVE2, as is the case between Cdc42 and WASP/N-WASP proteins. Based mostly on in vitro studies, IRSp53 was reported to be an important candidate linking WAVE2 to Rac1 (Miki and Takenawa, 2002;Miki et al, 2000;Oda et al, 2005;Suetsugu et al, 2006a). However, whether IRSp53 functions as an essential intermediate between Rac1 and WAVE2 in vivo was not established.…”