CrkL is an adapter for Wiskott-Aldrich syndrome protein and Syk

Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia are caused by mutations of the WAS protein (WASP) gene. WASP may be involved in the regulation of podosome, an actin-rich dynamic cell adhesion structure formed by various types of cells. The molecular links between WASP and podosomes or other cell adhesion structures are unknown. Platelets express an SH2-SH3 adapter molecule, CrkL, that can directly associate with paxillin, which is localized in podosomes. The hypothesis that CrkL binds to WASP was, therefore, tested. Results from coprecipitation experiments using anti-CrkL and GST-fusion proteins suggest that CrkL binds to WASP through its SH3 domain and that the binding was not affected by WASP tyrosine phosphorylation. The binding of GST-fusion SH3 domain of PSTPIP1 in vitro was also not affected by WASP tyrosine phosphorylation, suggesting that the binding of the SH3 domains to WASP is not inhibited by tyrosine phosphorylation of WASP. Anti-CrkL also coprecipitates a 72-kd protein, which was identified as syk tyrosine kinase, critical for collagen induced-platelet activation. CrkL immunoprecipitates contain kinase-active syk, as evidenced by an in vitro kinase assay. Coprecipitation experiments using GST-fusion CrkL proteins suggest that both SH2 and SH3 domains of CrkL are involved in the binding of CrkL to syk. WASP, CrkL, syk, and paxillin-like Hic-5 incorporated to platelet cytoskeleton after platelet aggregation. Thus, CrkL is a novel molecular adapter for WASP and syk and may potentially transfer these molecules to the cytoskeleton through association with cytoskeletal proteins such as Hic-5.     

All-trans-retinoic acid induces tyrosine phosphorylation of the CrkL adapter in acute promyelocytic leukemia cells

OBJECTIVE: All-trans-retinoic acid (RA) is a potent inducer of differentiation of acute promyelocytic leukemia (APL) cells in vitro and in vivo. It also exhibits synergistic effects with interferons on the induction of differentiation and growth inhibition in vitro. Recent studies showed that interferons engage a signaling pathway involving the CBL proto-oncogene and the CrkL adapter, which mediates interferon-induced growth inhibitory signals. The objective of this study was to determine whether the CBL-CrkL pathway is activated by treatment of the NB-4 and HL-60 acute leukemia cell lines with RA. MATERIALS AND METHODS: The effects of RA treatment on CBL and CrkL phosphorylation, as well as on protein-protein interactions, were determined in studies involving immunoprecipitations of cell extracts with specific antibodies and Western blots. In addition, glutathione-S-transferase fusion proteins were used in binding studies to determine whether the SH2 domain of CrkL interacts with CBL in a RA-dependent manner and whether Rapl is activated by RA. RESULTS: Treatment of NB-4 or HL-60 cells with RA resulted in strong tyrosine phosphorylation of CBL, which was time and dose dependent. Similarly, RA induced tyrosine phosphorylation of the CrkL adapter and the association of CrkL with CBL. The RA-dependent interaction of CrkL with CBL was mediated by binding of the SH2 domain of CrkL to tyrosine phosphorylated CBL, suggesting that CBL provides a docking site for engagement of CrkL in a RA-activated cellular pathway. The guanine exchange factor C3G was found to be associated with CrkL at similar levels before and after RA treatment, but Rapl activation downstream of C3G was not inducible by RA. CONCLUSIONS: These findings demonstrate that the CBL-CrkL pathway is one of the mediators of the effects of RA on APL cells and suggest that one of the mechanisms of synergy between RA and interferons may involve regulation of components of this signaling cascade.     

Adaptor proteins CRK and CRKL associate with the serine/threonine protein kinase GCKR promoting GCKR and SAPK activation

STE20-related kinases play significant regulatory roles in a range of cellular responses to environmental stimuli. GCKR (also referred to as KHS1) is a serine/threonine protein kinase that has an STE20-like protein kinase domain and that stimulates the stress-activated protein kinase (SAPK, also referred to as Jun kinase or JNK) pathway. GCKR has a large C-terminal regulatory domain that provides sites for interactions with other proteins. Adaptor proteins mediate the interactions between signaling molecules. In this study we showed that the adaptor proteins Crk and CrkL associated with GCKR. When Crk-I, Crk-II, or CrkL was transiently expressed in HEK 293T cells along with GCKR, each coimmunoprecipitated with GCKR. Furthermore, in the Bcr-Abl transformed cell line, K562 endogenous GCKR and CrkL coimmunoprecipitated, indicating a constitutive association. Detection of the CrkL-GCKR interaction required the SH3 domains of CrkL and 2 regions in GCKR-1 between amino acids 387 and 395 that contains a consensus SH3 binding motif and the other between amino acids 599 and 696. Crk or CrkL overexpression increased GCKR catalytic activity. A dominant negative form of Ras abolished Crk- or CrkL-induced GCKR activation, suggesting a dependence on Ras activation for their activation of GCKR. Finally, we showed impairment of the known ability of CrkL to activate the SAPK pathway by a catalytically inactive form of GCKR or by a GCKR antisense construct. Thus, GCKR associates with other proteins through interactions mediated by SH2/SH3 adaptor proteins, which can lead to GCKR and SAPK activation.     

CrkL and CrkII participate in the generation of the growth inhibitory effects of interferons on primary hematopoietic progenitors.

Interferons are potent regulators of normal and malignant hematopoietic cell proliferation in vitro and in vivo, but the signaling mechanisms by which they exhibit their growth inhibitory effects are unknown. We have recently shown that CrkL is engaged in Type I IFN signaling, as shown by its rapid tyrosine phosphorylation during engagement of the Type I IFN receptor. In the present study, we provide evidence that the related CrkII protein is also rapidly phosphorylated on tyrosine during treatment of U-266 and Daudi cells with IFNalpha or IFNbeta. We also show that both members of the Crk-family, CrkL and CrkII, are phosphorylated in an interferon-dependent manner in primary hematopoietic progenitors. Furthermore, inhibition of CrkL or CrkII protein expression by antisense oligonucleotides, reverses the inhibitory effects of IFNalpha or IFNgamma on the proliferation of normal bone marrow progenitor cells (colony forming units-granulocytic/monocytic [CFU-GM] and burst-forming units-erythroid [BFU-E]). Thus, both CrkL and CrkII are engaged in a signaling pathway (s) that mediates interferon-regulated inhibition of hematopoietic cell proliferation.     

Engagement of the CrkL adaptor in interferon alpha signalling in BCR-ABL-expressing cells

Interferon alpha (IFNalpha) has significant clinical activity in the treatment of patients with chronic myelogenous leukaemia (CML), but the mechanisms of its selective efficacy in the treatment of the disease are unknown. The CrkL adaptor protein interacts directly with the BCR-ABL fusion protein that causes the malignant transformation and is constitutively phosphorylated in BCR-ABL-expressing cells. In the present study, we provide evidence that CrkL was engaged in IFNalpha-signalling in the CML-derived KT-1 cell line, which expresses BCR-ABL and is sensitive to the growth inhibitory effects of IFNalpha. CrkL is constitutively associated with BCR-ABL in these cells and treatment with IFNalpha had no effect on the BCR-ABL/CrkL interaction. After IFNalpha stimulation, CrkL associated with Stat5, which also underwent phosphorylation in an IFNalpha-dependent manner. The interaction of CrkL with Stat5 was facilitated by the function of both the SH2 and the N-terminus SH3 domains of CrkL. The resulting CrkL-Stat5 complex translocated to the nucleus and could be detected in gel shift assays using elements derived from either the beta-casein promoter or the promoter of the PML gene, an IFNalpha-inducible gene that mediates growth inhibitory responses. In addition to its interaction with Stat5, CrkL interacts with C3G in KT-1 cells and such an interaction regulates the downstream activation of the small GTPase Rap1, which also mediates inhibition of cell proliferation. Thus, despite its engagement by BCR-ABL in CML-derived cells, CrkL mediates activation of downstream signalling pathways in response to the activated type I IFN receptor and such signals may contribute to the generation of the anti-proliferative effects of IFNalpha in CML.     

Characterization of Sam68-like mammalian proteins SLM-1 and SLM-2: SLM-1 is a Src substrate during mitosis

Sam68, the 68-kDa Src substrate associated during mitosis, is an RNA-binding protein with signaling properties that contains a GSG (GRP33, Sam68, GLD-1) domain. Here we report the cloning of two Sam68-like-mammalian proteins, SLM-1 and SLM-2. These proteins have an approximately 70% sequence identity with Sam68 in their GSG domain. SLM-1 and SLM-2 have the characteristic Sam68 SH2 and SH3 domain binding sites. SLM-1 is an RNA-binding protein that is tyrosine phosphorylated by Src during mitosis. SLM-1 bound the SH2 and SH3 domains of p59(fyn), Grb-2, phospholipase Cgamma-1 (PLCgamma-1), and/or p120(rasGAP), suggesting it may function as a multifunctional adapter protein for Src during mitosis. SLM-2 is an RNA-binding protein that is not tyrosine phosphorylated by Src or p59(fyn). Moreover, SLM-2 did not associate with the SH3 domains of p59(fyn), Grb-2, PLCgamma-1, or p120(rasGAP), suggesting that SLM-2 may not function as an adapter protein for these proteins. The identification of SLM-1 and SLM-2 demonstrates the presence of a Sam68/SLM family whose members have the potential to link signaling pathways with RNA metabolism.     

An unrecognized extracellular function for an intracellular adapter protein released from the cytoplasm into the tumor microenvironment

Mammalian cell membranes provide an interface between the intracellular and extracellular compartments. It is currently thought that cytoplasmic signaling adapter proteins play no functional role within the extracellular tumor environment. Here, by selecting combinatorial random peptide libraries in tumor-bearing mice, we uncovered a direct, specific, and functional interaction between CRKL, an adapter protein [with Src homology 2 (SH2)- and SH3-containing domains], and the plexin-semaphorin-integrin domain of β₁ integrin in the extracellular milieu. Through assays in vitro, in cellulo, and in vivo, we show that this unconventional and as yet unrecognized protein–protein interaction between a regulatory integrin domain (rather than a ligand-binding one) and an intracellular adapter (acting outside of the cells) triggers an alternative integrin-mediated cascade for cell growth and survival. Based on these data, here we propose that a secreted form of the SH3/SH2 adaptor protein CRKL may act as a growth-promoting factor driving tumorigenesis and may lead to the development of cancer therapeutics targeting secreted CRKL.     

Modification of Ser59 in the unique N-terminal region of tyrosine kinase p56lck regulates specificity of its Src homology 2 domain.

During T-cell activation, Ser59 in the unique N-terminal region of p56lck is phosphorylated. Mutation of Ser59 to Glu59 mimics Ser59 phosphorylation, and upon CD4 crosslinking, this mutant p56lck induces tyrosine phosphorylation of intracellular proteins distinct from those induced by wild-type p56lck. Mutant and wild-type p56lck have similar affinities for CD4 and similar kinase activities. In glutathione S-transferase fusion proteins, the p56lck Src homology 2 (SH2) domain with the SH3 domain and the unique N-terminal region (including Ser59) has a different binding specificity for phosphotyrosyl proteins than the SH2 domain alone. Either deletion of the unique N-terminal region or mutation of Ser59 to Glu59 in the fusion protein reverts the phosphotyrosyl protein binding specificity back to that of the SH2 domain alone. These results suggest that phosphorylation of Ser59 regulates the function of p56lck by controlling binding specificity of its SH2 domain.     

CrkII signals from epidermal growth factor receptor to Ras.

A rat fibroblast mutant defective in oncogenic transformation and signaling from epidermal growth factor receptor to Ras has been isolated. The mutant contains dominant negative-type point mutations in the C-terminal SH3 domain of one crkII gene. Among the adapters tested, the mutant is complemented only by crkII cDNA. Expression of the mutated crkII in parent cells generates the phenotype indistinguishable from the mutant cell. Yet overexpression or reduced expression of Grb2 in the mutant before and after complementation with crkII have little effect on its phenotype. We conclude that adapter molecules are highly specific and that the oncogenic growth signal from epidermal growth factor receptor to Ras is predominantly mediated by CrkII in rat fibroblast.     

Structure of a regulatory complex involving the Abl SH3 domain,the Crk SH2 domain,and a Crk-derived phosphopeptide

On phosphorylation of Y221 by Abelson (Abl) kinase, the Crk-II adapter protein undergoes an intramolecular reorganization initiated by the binding of its own Src homology 2 (SH2) domain to the pY221 site. Conformational changes induced by phosphotyrosine recognition promote the binding of the Src homology 3 (SH3) domain of the Abl tyrosine kinase to a proline-rich loop located between the betaD and betaE strands of the SH2 domain (DE loop). We have determined the NMR solution structure of the ternary complex of the Abl SH3 domain with the Crk SH2 domain bound to a Crk pY221 phosphopeptide. The SH2 domain bridges two ligands that bind at distinct sites. The interaction between the Abl SH3 domain and the Crk SH2 domain is localized to a canonical eight-residue site within the DE loop. From (15)N relaxation experiments, the DE loop of the SH2 domain in the complex displays a significant degree of conformational freedom. The structural and dynamic data therefore indicate that these SH2 and SH3 domains do not assume a unique orientation with respect to one another; rather, they appear to be only tethered via the DE loop. Thus, SH2 domain-SH3 domain interactions do not require additional tertiary contacts or restriction of domain orientation when a recognition motif is presented in a mobile loop. This complex between the Abl SH3 domain, Crk SH2 domain, and Crk phosphopeptide is an example of the extremely modular nature of regulatory proteins that provides a rich repertoire of mechanisms for control of biological function.     

DOCK2 associates with CrkL and regulates Rac1 in human leukemia cell lines

The CDM (ced-5 of Caenorhabditis elegans, DOCK180 [downstream of Crk with molecular weight of 180 kDa] of humans, and myoblast city of Drosophila melanogaster) family of proteins has been shown to play a pivotal role in the integrin-mediated signaling pathway under the regulation of an adaptor molecule c-CT10-related kinase II (c-Crk-II) in adherent cells. Recently, hematopoietic cell-specific CDM protein DOCK2 has been shown to be indispensable for lymphocyte migration. However, the regulatory mechanism for DOCK2 is still unknown because DOCK2 lacks a c-Crk-II binding consensus motif. In this study, we demonstrated that DOCK2 bound to CrkL, which is present exclusively in hematopoietic cells both in vivo and in vitro, and we also found that 2 separate regions of DOCK2 contributed to its binding to Src homology 3 (SH3) domain of CrkL. Colocalization of DOCK2 with Crk-like (CrkL) and F-actin was shown by immunocytochemical analysis with the use of Jurkat cells. We also found that CrkL-induced activation of small guanine triphosphatase (GTPase) Rac1 was significantly inhibited by the DOCK2-dCS mutant in 293T cells. Furthermore, the association of DOCK2 and Vav, the guanine-nucleotide exchanging factor (GEF) for Rac1, was demonstrated in Jurkat cells. Finally, the stable expression of DOCK2-dCS mutant in Jurkat cells was shown to reduce cell attachment. These data suggest the presence of a novel protein complex of CrkL, DOCK2, and Vav to regulate Rac1 in leukemia cell lines.     

SLAP, a dimeric adapter protein, plays a functional role in T cell receptor signaling

Engagement of the T cell antigen receptor (TCR) leads to rapid activation of protein tyrosine kinases, which in turn phosphorylate downstream enzymes and adapter proteins. Some adapter proteins, such as SLP-76, Vav, and LAT, positively regulate TCR-mediated signal transduction, whereas others, such as Cbl, play an inhibitory role. SLAP (Src-like adapter protein), an adapter protein containing a Src homology 3 and a Src homology 2 domain, was isolated from a yeast interacting screen by using N-terminal Cbl as bait. N-terminal Cbl interacts with SLAP in vivo and in vitro in a tyrosine phosphorylation-independent manner. We observed that SLAP is expressed in T cells, and upon TCR activation, SLAP interacts with ZAP-70, Syk, LAT, and TCRzeta chain in Jurkat T cells. In transiently transfected COS-7 cells, SLAP forms separate complexes with ZAP-70, Syk, and LAT through its Src homology 2 domain. Overexpression of a C-terminal-truncated SLAP mutant down-regulates nuclear factor of activated T cells-AP1 activity. We have evidence that SLAP forms homodimers through its C-terminal region. Serial truncations and mutations in the C terminus of SLAP demonstrate that there is a correlation between the loss of dimerization and the inhibition of nuclear factor of activated T cells-AP1 activity. The in vivo association of SLAP with key signaling molecules and its inhibition of T cell activation suggests that SLAP plays an important role in TCR-mediated signal transduction.     

The infected cell protein 0 of herpes simplex virus 1 dynamically interacts with proteasomes, binds and activates the cdc34 E2 ubiquitin-conjugating enzyme, and possesses in vitro E3 ubiquitin ligase activity

The infected cell protein 0 (ICP0) of herpes simplex virus 1, a promiscuous transactivator shown to enhance the expression of genes introduced into cells by infection or transfection, interacts with numerous cellular proteins and has been linked to the disruption of ND10 and degradation of several proteins. ICP0 contains a RING finger domain characteristic of a class of E3 ubiquitin ligases. We report that: (i) in infected cells, ICP0 interacts dynamically with proteasomes and is bound to proteasomes in the presence of the proteasome inhibitor MG132. Also in infected cells, cdc34, a polyubiquitinated E2 ubiquitin-conjugating enzyme, exhibits increased ICP0-dependent dynamic interaction with proteasomes. (ii) In an in vitro substrate-independent ubiquitination system, the RING finger domain encoded by exon 2 of ICP0 binds cdc34, whereas the carboxyl-terminal domain of ICP0 functions as an E3 ligase independent of the RING finger domain. The results indicate that ICP0 can act as a unimolecular E3 ubiquitin ligase and that it promotes ubiquitin-protein ligation and binds the E2 cdc34. It differs from other unimolecular E3 ligases in that the domain containing the RING finger binds E2, whereas the ligase activity maps to a different domain of the protein. The results also suggest that ICP0 shuttles between nucleus and cytoplasm as a function of its dynamic interactions with proteasomes.     

Domain requirements for the Dock adapter protein in growth- cone signaling

Tyrosine phosphorylation has been implicated in growth-cone guidance through genetic, biochemical, and pharmacological studies. Adapter proteins containing src homology 2 (SH2) domains and src homology 3 (SH3) domains provide a means of linking guidance signaling through phosphotyrosine to downstream effectors regulating growth-cone motility. The Drosophila adapter, Dreadlocks (Dock), the homolog of mammalian Nck containing three N-terminal SH3 domains and a single SH2 domain, is highly specialized for growth-cone guidance. In this paper, we demonstrate that Dock can couple signals in either an SH2-dependent or an SH2-independent fashion in photoreceptor (R cell) growth cones, and that Dock displays different domain requirements in different neurons.     

Platelet activation via the collagen receptor GPVI is not altered in platelets from chronic myeloid leukaemia patients despite the presence of the constitutively phosphorylated adapter protein CrkL

In this study, we show that the adapter proteins CrkL and Cbl undergo increases in tyrosine phosphorylation and form an intracellular complex in platelets stimulated with the snake venom toxin convulxin, a selective agonist at the collagen receptor glycoprotein VI (GPVI). Constitutive tyrosine phosphorylation of CrkL has previously been reported in platelets from chronic myeloid leukaemia (CML) patients. This was confirmed in the present study, and shown to result in a weak constitutive association of CrkL with Cbl and a number of other unidentified tyrosine-phosphorylated proteins. There was no further increase in phosphorylation of CrkL in CML platelets in response to GPVI activation, whereas phosphorylation of Cbl and its association with CrkL were potentiated. In addition, this was accompanied by a small increase in p42/ 44 mapkinase (MAPK) activity in CML platelets. The functional consequence of the presence of constitutively phosphorylated proteins in CML platelets was investigated by measurement of aminophospholipid exposure and alpha-granule secretion. This revealed little alteration in the concentration-response curves for either in CML platelets stimulated via GPVI, although maximal levels of P-selectin were depressed. Despite the minimal effect on platelet activation in CML patients, we cannot exclude a role for CrkL or Cbl in signal transduction pathways stimulated via GPVI.     

Stem cell factor induces phosphatidylinositol 3'-kinase-dependent Lyn/Tec/Dok-1 complex formation in hematopoietic cells

Stem cell factor (SCF) has an important role in the proliferation, differentiation, survival, and migration of hematopoietic cells. SCF exerts its effects by binding to cKit, a receptor with intrinsic tyrosine kinase activity. Activation of phosphatidylinositol 3'-kinase (PI3-K) by cKit was previously shown to contribute to many SCF-induced cellular responses. Therefore, PI3-K-dependent signaling pathways activated by SCF were investigated. The PI3-K-dependent activation and phosphorylation of the tyrosine kinase Tec and the adapter molecule p62Dok-1 are reported. The study shows that Tec and Dok-1 form a stable complex with Lyn and 2 unidentified phosphoproteins of 56 and 140 kd. Both the Tec homology and the SH2 domain of Tec were identified as being required for the interaction with Dok-1, whereas 2 domains in Dok-1 appeared to mediate the association with Tec. In addition, Tec and Lyn were shown to phosphorylate Dok-1, whereas phosphorylated Dok-1 was demonstrated to bind to the SH2 domains of several signaling molecules activated by SCF, including Abl, CrkL, SHIP, and PLCgamma-1, but not those of Vav and Shc. These findings suggest that p62Dok-1 may function as an important scaffold molecule in cKit-mediated signaling.