Dok-R Binds c-Abl and Regulates Abl Kinase Activity and Mediates Cytoskeletal Reorganization

Abstract

Dok-R, also known as Dok-2/FRIP, belongs to the DOK family of signaling molecules that become tyrosinephosphorylated by several different receptor and cytoplasmic tyrosine kinases. Tyrosine phosphorylation of DOK proteins establishes high affinity binding sites for other signaling molecules leading to activation of a signaling cascade. Here we show that Dok-R associates with c-Abl directly via a constitutive SH3-mediated interaction and that this binding requires a PMMP motif in the proline-rich tail of Dok-R. The Dok-R-Abl interaction is further enhanced by an active c-Abl kinase, which requires the presence of its SH2 domain. Interaction of Dok-R with c-Abl also results in an increase in c-Abl tyrosine phosphorylation and kinase activity. Furthermore, we demonstrate that this increase in kinase activity correlates with a concomitant increase in c-Ablmediated biological activity as measured by the formation of actin microspikes. Our data are the first to demonstrate that Dok-R and c-Abl interact in both a constitutive and inducible fashion and that Dok-R influences the intracellular kinase and biological activity of c-Abl. Activation of receptor and cytoplasmic tyrosine kinases (TK)1 induces tyrosine phosphorylation of target proteins that initiates a series of protein-protein interactions (1). One of the many targets of TK-mediated signaling pathways is the actin cytoskeleton (2). Actin cytoskeletal rearrangements are important fundamental events that occur during a variety of cellular processes such as adhesion, spreading, migration, proliferation, and differentiation (2). Reorganization of the actin cytoskeleton involves cross-talk between a multitude of different signaling pathways involving kinases, integrins, and other adhesion molecules that target the actin cytoskeletal machinery. Many of these signaling proteins, including the integrin receptors and the cytosolic c-Abl tyrosine kinase, associate directly with the cytoskeleton.Tyrosine phosphorylation of docking and adapter proteins by receptor and cytosolic TKs promotes signal amplification by providing high affinity binding sites for numerous intracellular signaling molecules containing Src homology 2 (SH2) domains or phosphotyrosine binding (PTB) domains. Protein-protein interactions can also occur in a non-inducible manner as seen with the constitutive interaction between Src homology 3 (SH3) domains binding proline-rich motifs. Pleckstrin homology (PH) domains on signaling proteins recognize phospholipids and assist in recruitment of the protein to the plasma membrane. In addition to their role in mediating protein interactions, many of these modular domains found in cytoplasmic TKs also serve to regulate the host kinase.Similar to Src family kinases, c-Abl kinase activity is tightly regulated through intramolecular interactions involving its amino-terminal SH3 domain-containing region (3-5) and can be activated by epidermal and platelet derived growth factors in addition to Src and Src-like kinases (6). c-Abl contains a large array of functional d...

c-Abl Expression in Chronic Lymphocytic Leukemia Cells: Clinical and Therapeutic Implications

“…Because c-Abl is implicated in B-cell receptor signaling, this kinase could then, in turn, be at least partly responsible for some of the effects of this signaling. If so, our experiments showing that STI-571 inhibits c-Abl-induced phosphorylation of DOK2, a known c-Abl substrate (23), support the proposition that, in unmutated CLL, signals from the B-cell receptor provide cell rescue from apoptosis (1,27). Such rescue includes an activation of NF-nB that partly depends on constitutively active c-Abl.…”

“…This shows that STI-571 was an effective inhibitor of c-Abl in intact cells. DOK2 is a known substrate of active c-Abl in hemopoietic cells (23) and its phosphorylation on tyrosine was used as a measure of c-Abl activity in KCL22 and CLL cells. Figure 4B shows that treatment of these cells with 10 Amol/L STI-571 markedly reduced the phosphorylation of DOK2, suggesting that STI-571 also inhibits c-Abl activity in intact CLL cells.…”

The R-Ras interaction partner ORP3 regulates cell adhesion

“…Protrusion formation similar to that induced by ORP3 has been reported to occur upon overexpression of several other proteins, all of which represent regulators of the actin cytoskeleton: Cdc42 effector protein 1 (CEP1/MSE55) (Burbelo et al, 1999), ATPbinding cassette transporter-1 (ABCA1) (Wang, N. et al, 2000;Tsukamoto et al, 2001), calpain 2 (CAPN2) (Franco et al, 2004;Perrin et al, 2006), downstream of kinase 2 (Dok-2, Dok-R or FRIP) (Master et al, 2003) and c-Abl tyrosine kinase (Master et al, 2003). Dok-2 and c-Abl represent proteins associated with Nck (Master et al, 2001;Master et al, 2003), which is involved in the regulation of R-Ras function (Wang, B. et al, 2000) and the actin cytoskeleton (Buday et al, 2002).…”

“…Dok-2 and c-Abl represent proteins associated with Nck (Master et al, 2001;Master et al, 2003), which is involved in the regulation of R-Ras function (Wang, B. et al, 2000) and the actin cytoskeleton (Buday et al, 2002). Nck binds to Wiskott-Aldrich syndrome protein (WASP), which controls the nucleation of actin filaments by the Arp2/3 complex (Buday et al, 2002).…”

3BP2-deficient mice are osteoporotic with impaired osteoblast and osteoclast functions

“…Abl exists in an inactive autoinhibited state in which the SH3 and SH2 domains bind through intramolecular interactions to the kinase domain and function as a "clamp" to maintain a conformer of low catalytic activity (37). Release of the SH3-SH2 domain from the kinase domain through competing occupancy of the SH3 and SH2 domains by cognate ligands in-trans results in efficient activation of Abl (38)(39)(40). Phosphorylation of tyrosine 412 (Y 412 ) (41) in the activation helix and tyrosine 245 (Y 245 ) (42) in the kinase-SH2 linker sequence were associated with a state of high Abl kinase activity.…”