The Product of the cbl Oncogene Forms Stable Complexes In Vivo with Endogenous Crk in a Tyrosine Phosphorylation-Dependent Manner

Abstract

The Crk proteins, originally isolated as an oncogene product in an avian sarcoma virus (26), belong to a class of adapter proteins containing Src homology 2 (SH2) and SH3 domains. Other members of this group include Grb2/ASH (11) and Nck (13). These bifunctional proteins are thought to couple tyrosine-phosphorylated receptors or their substrates via the SH2 domain to downstream effectors via the SH3 domain. Three cellular homologs of v-Crk have been identified: Crk-I, a 21-kDa protein with only one SH2 and one SH3 domain (24); Crk-II, 40-and 42-kDa proteins consisting of one SH2 and two SH3 domains (36); and CrkL, a 36-kDa Crk-like protein with one SH2 and two SH3 domains (49). Despite their lack of demonstrated enzymatic activity, expression of v-Crk or Crk-I but not Crk-II leads to cell transformation and increased tyrosine phosphorylation (24,27). Furthermore, v-Crk binds directly to the major tyrosine-phosphorylated proteins present in v-Crk-transformed cells, presumably via its SH2 domain (22, 23). These phosphoproteins include paxillin, a focal adhesionassociated protein (3), and the newly identified p130 cas (39). p130cas has no known catalytic domain but contains a cluster of predicted high-affinity Crk-SH2 binding motifs and forms a stable complex in vivo with v-Crk. The Crk-SH3 domain has been shown to interact with several proteins, including Abl (8), Sos (9, 21), C3G (9, 15, 21, 46), and, more recently, Eps15 (42). Since Sos and C3G contain a guanine nucleotide exchange activity, Crk proteins have been hypothesized to play a role in the regulation of p21 ras -GTP formation. Both the SH2 and SH3 domains of v-Crk are required for the transformation of chicken embryo fibroblasts (27). In addition, Crk mutants with dysfunctional SH2 or SH3 domains inhibited neuronal differentiation of PC-12 cells and nerve growth factor-induced activation of Ras (21, 45).The c-Abl oncogene product is a member of the nonreceptor class of tyrosine kinases. Like c-Crk-II, c-Abl overexpression does not lead to cellular transformation (10,14). However, the chromosomal translocation of c-abl to the bcr gene in Philadelphia chromosome-positive human leukemias produced the 210-and 190-kDa chimeric Bcr-Abl proteins (6, 16). These fusion proteins have elevated tyrosine kinase activity and transforming properties (20,32). Although the precise mechanisms involved in cellular transformation by Bcr-Abl remain unclear, several key signaling proteins are known to be tyrosine phosphorylated in cells bearing this oncogene, including Bcr-Abl itself (32) as well as Shc (35), CrkL (48), paxillin (40), and the proto-oncogene product of cbl (1). Moreover, activation of Ras has also been implicated in Bcr-Abl-mediated cellular transformation (34,35).To evaluate the potential role of Crk proteins in the transforming functions of Bcr-Abl, we have examined their interactions in the human chronic myelogenous leukemia cell line K562. We report here that, unlike observations in v-Crk-and v-Src-transformed cells, p130cas is not tyrosine phosphorylated...

PMA-induced phenotypic changes in K562 cells: MAPK-dependent and -independent events

“…We have previously shown that K562 cells contain tyrosine phosphorylated signaling proteins associated with the adapter proteins CrkII and CrkL. 39 As seen in Figure 2a, PMA induces a reduction in the tyrosine content or amount of a CrkLassociated 210 kDa protein and an increase in the tyrosine content or/amount of a CrkL-associated 60 kDa protein. The effect of PMA on the 210 kDa protein is independent of the MAPK pathway while the effect on the 60 kDa protein is dependent on this activity.…”

“…The behavior of the 210 kDa protein is consistent with that of the Bcr-Abl kinase. 27 The amount of tyrosine phosphorylated Cbl, a known partner of CrkL that is present in K562 cells, 39 remains unchanged (Figure 2b) although a slight increase in the total amount of c-Cbl protein under the conditions studied is seen (data not shown). The level of CrkL protein (Figure 2c) is not significantly changed by the culturing conditions although a noticeable change in the tyrosine content of CrkL was observed (Figure 2c).…”

Identification of Tyr-762 in the platelet-derived growth factor α-receptor as the binding site for Crk proteins

“…The presence of both SH2 and SH3 domains in Crk proteins is of crucial importance for their function as adaptor molecules (Tanaka et al, 1993(Tanaka et al, , 1995. The SH2 domains of Crk molecules have been shown to bind to several phosphorylated proteins, including p130Cas, Cbl and the focal adhesion protein paxillin Buday et al, 1996;Ribon et al, 1996;Sakai et al, 1994;Smit et al, 1996b). The SH3 domains have been shown to bind to e.g.…”

Tyrosine kinase activity of the EGF receptor is enhanced by the expression of oncogenic 70Z-Cbl

“…This suggests a pivotal role in early events following signal transduction across the cell membrane into the cytoplasm. Furthermore, Cbl has been found to associate constitutively or inductively with many signalling proteins such as the Grb2, Crk and Nck adaptor proteins, members of the Src, Abl and Syk tyrosine kinase families, the p85 regulatory subunit of PI 3-kinase and 14-3-3 proteins (Donovan et al, 1994;de Jong et al, 1995;Buday et al, 1996;RiveroLezcano et al, 1994;Ribon et al, 1996;Andoniou et al, 1994Andoniou et al, , 1996Smit et al, 1996;Tanaka et al, 1996;Tsygankov et al, 1996;Fournel et al, 1996;Kim et al, 1995;Meisner et al, 1995;Solto and Cantley, 1996;Liu et al, 1996). To date however a de®nite function for Cbl has not emerged from these studies.…”