Leukemia (1998) 12, 637–644  1998 Stockton Press All rights reserved 0887-6924/98 $12.00 http://www.stockton-press.co.uk/leu REVIEW

Role of the adapter CRKL in of normal hematopoietic and BCR/ABL-transformed cells M Sattler and R Salgia

Division of Hematologic Malignancies, Department of Adult Oncology, Dana-Farber Cancer Institute, 44 Binney St, Boston, MA 02115, USA

CRKL is a 39 kDa adapter protein, originally cloned in proximity CRK (‘CT10 regulator of kinase’) family, and transforms to the BCR on 22, which has a key regulat- chicken cells with increased of ory role in hematopoietic cells. CRKL has one SH2 and two SH3 8 domains, with 60% homology to CRK II. CRKL is a prominent cellular . The v-crk sequence encodes for a 47 kDa substrate of the BCR/ABL oncoprotein in chronic myelogenous Gag-Crk fusion protein, which has a deletion of the C-terminal leukemia and binds to both BCR/ABL and c-ABL. CRKL has SH3 domain of CRK.5,8 been shown to be tryosine phosphorylated in response to nor- mal hematopoietic receptor signaling with ligands such as thrombopoietin, erythropoietin or steel factor. CRKL functions as an adapter protein Additionally, CRKL is involved in signaling initiated by crosslinking of ␤ , and B cell or T cell receptors. Struc- turally, the amino-terminal SH3 domain of CRKL has been Like other SH2 and SH3 domain containing proteins such as shown to bind proteins such as C3G, SOS, PI3-K, c-ABL or NCK, GRB2, or even CRK, CRKL has been shown to function BCR/ABL. The SH2 domain of CRKL can bind to tyrosine phos- as an ‘adapter protein’, linking different proteins in a regulated phorylated proteins such as CBL, HEF1, CAS or . This manner in signaling. The known CRKL SH2 and SH3 binding review summarizes the current knowledge on the function of proteins are summarized in Figure 1. The specificity of an this unique adapter protein in normal hematopoietic and leu- kemic . adapter protein is defined through the structure and function Keywords: CRKL; CRK; BCR/ABL; signal transduction; hematopo- of SH2 and SH3 domains to recognize specific phosphotyros- ietic growth factor; ine and -rich motifs, respectively. By in vitro analysis, the CRK SH2 domain can ‘tail-back’ on itself by recognizing a YXXP motif (Tyr-X-X-Pro) intramolecularly;9 however, this Historical perspective has not been shown for the CRKL-SH2 domain. Tyrosine phosphorylation of cellular proteins is an CRKL was originally cloned by ten Hoeve et al1 in an effort important event in hematopoietic signaling. CRKL has been to identify located between the BCR gene and the cen- shown to be tyrosine phosphorylated in response to hemato- 10 tromere of , band q11. An exon in this region poietic growth factor stimulation, including thrombopoietin, 11 12 was found to encode a possible SH2 domain, and the com- erythropoietin, and steel factor. Also, CRKL binds consti- plete cDNA sequence revealed a novel CRK-like gene, termed tutively and inducibly to key molecules in cellular signaling CRKL. The CRKL gene encodes for a 39 kDa protein, and has such as in response to or hematopoietic growth factor an overall homology of 60% to c-CRK.1 Three independent crosslinking (Figure 2). In CML, CRKL is tyrosine phosphoryl- laboratories published, in 1994,2–4 an exciting and novel ated and interacts directly with the transforming protein 2–4 observation that CRKL is a major tyrosine phosphorylated pro- BCR/ABL. The function of CRKL tyrosine phosphorylation tein in primary cells or cell lines derived from patients with in normal and leukemic cell signaling is not known. It is poss- chronic myelogenous leukemia (CML). Since then, CRKL has ible that tyrosine phosphorylation may help CRKL to function been shown to play an important role in normal hemato- as a signaling molecule. Various interactions of CRKL occur poietic cell signaling as well as BCR/ABL transformation. through SH domains, and the current knowledge about the role of CRKL as it interacts with other proteins in normal and leukemic cell signaling is discussed below. CRKL is a member of the CRK family proteins

The family of CRK proteins has been defined to include CRKL, The SH3 domains of CRKL in normal cell signaling c-CRK, and v-CRK (Figure 1). c-CRK is the human homolog of the v-Crk oncogene,5 and is localized on chromosome 17.6 SH3 domains are comprised of about 60 amino acids and Both CRKL and c-CRK are ubiquitously expressed proteins mediated protein–protein interactions with proline-rich motifs with one SH2 domain, an SH2′ domain (a putative SH2 of the general sequence PXXP (Pro-X-X-Pro; X indicates no domain with no known function), and two SH3 domains.1,5 specific ). The proline-rich motifs are located in a In normal cells, c-CRK is mainly expressed as CRK-II helical structure and bind mostly through hydrophobic inter- (containing both SH3 domains and hereafter referred as CRK), actions to the SH3 binding pocket. SH3 domains recognize and to a lesser extent, as CRK-I (only containing the N- proline-rich helices in forward and reverse configuration. The terminal SH3 domain).7 v-Crk, the transforming protein in the two possible different configurations in the SH3 target protein avian retrovirus CT10, was the original cloned member of the have the consensus sequence of XpXPpXP (X-p-X-Pro-p-X- Pro = class I; p indicates a residue that tends to be proline) or XPpXPpX (X-Pro-p-X-Pro-p-X = class II), respectively. The two Correspondence: M Sattler or R Salgia; Fax: 617 632 4388 critical conserved are located in two different hydro- Received 3 September 1997; accepted 26 January 1998 phobic pockets, in part formed by aromatic residues in the Review M Sattler and R Salgia 638

Figure 1 Graphic representation of CRK family members and CRKL binding proteins. SH, src homology domain; DH, dbl homology domain; PH, pleckstrin homology domain; cdc25-l, cdc25-like domain; NLS, nuclear localization sequence; DBD, DNA binding domain; ABD, binding domain; RF, ring finger; LZ, zipper; LIM, LIM domain; P, proline; Y, tyrosine. Review M Sattler and R Salgia 639 Table 1 Summary of CRK and CRKL SH2/SH3 domain binding C3G, SOS proteins The understanding of CRKL function can be partially obtained CRK CRKL Ref. through its binding partners. For example, in B cells and T cells CRKL is constitutively linked to the nucleotide exchange SH3 binding protein 19,20,34,35 C3G ++ 17–20 factors C3G and SOS (Figure 4). SOS has known RAS SOS ++17,20,21 guanine exchange factor activity for p21 ; whereas C3G PI3-K ND + 12 appears to have specific guanine exchange activity for c-ABL ++ 9,20,21 p21RAP1.36 C3G is a member of the SOS family with similarity BCR/ABL ++ 20–22 to the protein tyrosine phosphatase CDC25.37 C3G does not ARG + ND 23 RAS 36 RAP1 + have substrate specificity for p21 , but its substrate p21 DOCK ND 24 RAS EPS/EPSR + ND 25 appears to regulate, at least in part, the signal from p21 to the RAF kinase. The CRKL/C3G complex has been found to SH2 binding protein be linked to tyrosine phosphorylated proteins, such as CBL in CBL ++18,22,26 T cell signaling.19 In anergic T cells, which have been found CAS ++ 27,28 to have elevated levels of active p21RAP1, there is a constitutive + HEF1 ND 29–31 complex of CRKL with C3G and CBL.38 paxillin ++ 32,33

+, known interaction; ND, interaction not determined. PI3-K

In signaling through c-KIT, the receptor for steel factor, CRKL SH3 domain.13,14 Recently a consensus sequence for binding has been described to be directly downstream of the c- to the CRK SH3 domain, PXLPXK (Pro-X-Leu-Pro-X-Lys), has KIT/PI3-K receptor complex (Figure 2). After steel factor stimu- been described.15,16 It is likely that the CRKL binding domains lation in the megakaryoblastic MO7e cell line, CRKL was in recognize the same or similar motifs/proteins as CRK (Table 1). a stable complex that contained c-KIT and PI3-K Consistent with the structural and functional homology, all (phosphatidylinositol 3-kinase). The interaction of CRKL with known CRKL SH3 binding proteins, including C3G, SOS, PI3- the 85 kDa regulatory subunit of PI3-K was inducible. The K and c-ABL contain this motif (Figure 3). Also, it has been CRKL interaction with the 85 kDa regulatory subunit was shown that the SH3 domain of CRK proteins involved in bind- mediated via the CRKL-SH3 domain.12 The biological effects ing other proteins is likely to be the amino-terminal SH3 of activated PI3-K may vary widely from cell to cell, ranging domain but not the carboxy-terminal SH3 domain.17 from regulation of apoptosis, viability, or early gene Described below are some proteins that are known to interact expression, to regulation of adhesion.39–41 Interestingly, recent through the CRKL SH3 domain. findings suggest that PI3-K activates the kinase AKT

Figure 2 Model of CRKL in a stable complex with CBL and PI3-K after steel factor (SF) stimulation and in BCR/ABL-transformed cells. PI3-K can activate the serine kinase AKT which in turn inactivates BAD. BAD phosphorylated on serine residues can bind to 14-3-3 proteins and is thus prevented from contributing to apoptosis. Review M Sattler and R Salgia 640

Figure 3 Sequence comparison of proline-rich domains of CRKL SH3 binding proteins (proper amino acid numbers shown) with the CRK SH3 consensus binding motif.

Figure 4 CRKL is partially localized to the cytoskeleton. A model of a with actin fibers leading to an integrin receptor ligated with proteins (ECM) is shown. The CRKL SH2 domain interacts with tyrosine phosphorylated CBL, CAS, HEF1, or paxillin, and the CRKL SH3 domain binds to C3G, SOS, or BCR/ABL. Direct interaction of CRKL with BCR/ABL contributes to transformation by this oncoprotein.

which in turn phosphorylates the BCL-2-related protein of the c-ABL tyrosine kinase.9,52 However, in contrast to CRK, BAD.42–45 If phosphorylated by AKT BAD can bind to 14-3-3 a role of c-ABL as the CRKL tyrosine kinase in normal cells proteins and its proapoptotic potential is blocked46 (Figure 2). has not yet been shown. Thus, the CRKL/PI3-K interaction may be quite important in It is not clear at this time if CRKL is a regulator of C3G, SOS, cell viability signaling. PI3-K, or c-ABL enzyme activity, or downstream effectors that require any of these enzyme activities for an as yet unknown signaling function. Nevertheless, CRKL is likely to act as an ABL intermediate that has the function to link the enzymes to tyro- sine phosphorylated proteins using the CRKL SH2 domain. The CRKL SH3 domain also binds constitutively to the tyrosine kinase c-ABL in hematopoietic and non-hematopoietic cells.21,22,47 However, a function of this CRKL/c-ABL signaling The SH2 domain of CRKL in normal cell signaling complex in normal signaling has yet to be described. c-ABL has been shown to be involved in transcriptional activation,48 SH2 domains are conserved phosphotyrosine binding motifs is activated in response to certain types of DNA damage,49,51 of approximately 100 amino acids. The specificity of SH2 and is mobilized in the cell in response to integrin stimu- domain interactions with phosphotyrosine within a lation.51 Interestingly, in v-Crk-transformed cells, several pro- substrate/target protein is mostly given by the second or third teins are heavily tyrosine phosphorylated, and it has been sug- amino acid carboxy terminal of this tyrosine residue. The con- gested that v-Crk and CRK may serve as regulatory subunits served amino acids in the SH2 binding pocket determine the Review M Sattler and R Salgia 641 specificity for phosphotyrosine binding, which occurs through though both cell lines express HEF1 and CBL.30 This provides hydrogen bonds and amino-aromatic interactions. Additional for the possibility of an intracellular signaling ‘switch’ that amino acids in the binding pocket determine the recognition could couple integrin signaling to different biological effects specificity of the amino acids adjacent to the phosphotyros- as related to CRKL. ine.53,54 Also, a point mutation of arginine (R) to lysine (K) in the conserved FLVRES (Phe-Leu-Val-Arg-Glu-Ser) motif in the SH2 domain interferes with phosphotyrosine binding.55,56 Role of CRKL in chronic myelogenous leukemia The CRK SH2 domain binds to pYXXP (phosphoTyr-X-X- Pro) motifs.57 Similarly, the CRKL SH2 domain has recently CML is caused by the Philadelphia chromosome translocation been shown to bind pYXXP motifs in the focal adhesion pro- (t[9;22]) which generates the potent oncogenic BCR/ABL tyro- tein paxillin33 and the proto-oncoprotein CBL.58 Also, the SH2 sine kinase. Clinically, patients with CML can have easy domain of CRKL has been found to bind to HEF1, or CAS.28,30 fatigue, abdominal complaints secondary to splenomegaly, It has been shown that the CRK-SH2 domain can intramolecu- leukocytosis, and anemia. CML is characterized by a stable larly bind to the tyrosine at position 221(Y221XXP).9,59 Simi- phase in which there is excessive proliferation and accumu- larly, it has been hypothesized that the CRKL-SH2 domain can lation of cell in the myeloid series. In the stable phase, the potentially bind the tyrosine at position 207 (Y207XXP), how- leukocyte alkaline phosphatase score is low or absent in over ever, this has not yet been shown. Binding of CRKL Y207 or 90% of patients with CML. Stable phase invariably can lead CRK Y221 by its own SH2 domains would preclude binding to progression with an advanced leukemic phase. Typically, by other target proteins. the median interval is 3.5–5 years for conversion from chronic to blastic phase. The prognosis of patients with blastic phase leukemia is quite poor.71–73 CBL There are multiple substrates for tyrosine phosphorylation in CML cells. CRKL was shown to be a prominent tyrosine One major CRKL SH2 binding protein is identified as the phosphorylated protein in primary cells from patients with 120 kDa proto-oncoprotein CBL (for Casitas B lineage CML.2–4 BCR/ABL is localized to the cytoskeleton and induces lymphoma). The widely expressed CBL protein is tyrosine tyrosine phosphorylation of multiple substrates, including phosphorylated and associates with CRKL after growth factor BCR/ABL itself, CRKL, CBL, HEF1, CAS, paxillin, and other receptor,11,12 integrin receptor,30 or in anergic T cells.38 For proteins.73 As a distinct difference to CRKL, CRK has not been example, CBL is tyrosine phosphorylated after steel factor shown to be tyrosine phosphorylated in BCR/ABL-transformed stimulation or ␤1 and ␤2 integrin ligation in MO7e cells. Nevertheless, tyrosine phosphorylation of CRKL does cells.12,30,60 Also, ␤1 integrin ligation or B cell receptor lig- not appear to be important for transformation by BCR/ABL ation in human B cells stimulates CBL tyrosine phosphoryl- since overexpression of the Y207 to Phe CRKL mutant in ation.61 CBL is the cellular homolog of v-Cbl, the oncoprotein myeloid cells did not effect BCR/ABL transformation. It should in the CAS NS-1 retrovirus which induces pre-B cell lym- be also noted that phosphorylation of Y207 in CRKL has not phomas and myelogenous leukemias in mice.62–64 In mam- been found to be important for fomation of signaling com- malian cells, the specific function of CBL is not known, but plexes.74 Overexpression of CRKL in Rat-1 fibroblasts but not the CBL homolog Sli-1 in C. elegans is a negative regulator of hematopoietic cells can lead to transformation and activation the epidermal growth factor receptor tyrosine kinase homo- of the RAS pathway.47,75 It is believed that in CML CRKL links logue Let-23.65 Also, CBL-B,aCBL homolog with 50% several key molecules leading to leukemic cell growth and/or sequence similarity has been recently cloned,66 and it will be inhibition of apoptosis. interesting if this can interact with CRKL in hematopoietic cells. CRKL is linked to BCR/ABL in CML

CAS, HEF1 BCR/ABL interacts through a proline-rich sequence within ABL directly with the CRKL SH3 domain22,76 (Figure 2). CRKL The interaction of the CRKL SH2 domain with its target pro- becomes tyrosine phosphorylated by BCR/ABL on tyrosine teins has been studied in integrin signaling. Ligation of ␤1 207 Y207,74 binds with its SH2 domain to CBL and is in a integrin receptors induces association of CRKL with CBL, multimeric complex with PI3-K, c-ABL, and BCR/ABL.22,26 It HEF1, or CAS.29,30 CAS (for Crk-associated substrate) was is likely that CRKL SH2 binding to tyrosine phosphorylated cloned as a major 130 kDa tyrosine phosphorylated protein proteins also leads to activation of other pathways that could in response to v-crk transformation.27 Currently, there are contribute to leukemic transformation. Interestingly, binding three CAS-related molecules (the 130 kDa protein CAS, the of CRKL through its SH3 to BCR/ABL appears to contribute to 110 kDa protein HEF1/CAS-L,67,68 and the 95 kDa protein leukemia, since deletion of the proline-rich domain that binds SIN/EFS69,70). The structure for CAS-family members includes CRKL in BCR/ABL reduces the ability of the oncoprotein to an amino-terminal SH3 domain, multiple YXXP motifs transform Rat-1 fibroblasts.47 In myeloid cells BCR/ABL leads (substrate domain), and potential SRC-binding sites at the C- to growth factor independence.77 Myeloid cell expressing terminus. The 110 kDa protein HEF1 (for human enhancer of BCR/ABL lacking the CRKL SH3 binding site are still growth filamentation 1) is a tissue-specific protein first identified dur- factor dependent.76 It is possible that transformation and ing cloning of human genes which induce morphological growth factor independence in myeloid cells is a different changes in S. cerevisiae. Expression of the HEF1 C-terminus regulated process than in fibroblasts. induces pseudohyphae in S. cerevisiae.67 Interestingly, in the Similar to normal cell signaling, the CRKL SH2 domain human megakaryoblastic cell line MO7e, CRKL was induced binds to CAS family proteins28 and CBL,22,26 and the interac- to bind to only CBL, and in the T cell line H9 CRKL was found tion of the known SH3 binding proteins C3G, SOS and ABL to bind to only HEF1 after ␤1 integrin cross-linking, even does not change upon BCR/ABL transformation.20 The contri- Review M Sattler and R Salgia 642 bution of CRKL to transformation is independent from GRB2, in several different signaling pathways. It is possible that the however, the exact mechanism is unknown.47 GRB2 is an biological functions of CRKL may also vary widely in different SH2-SH3-SH3 adapter protein that contributes to BCR/ABL cells. Since CRKL has a high homology to CRK, it will be of transformation by linking SOS, and thus the RAS-pathway, to special interest to compare the differences in tyrosine phos- Y177 in BCR/ABL.78,79 It is possible that multiple abnormalities phorylation and potential signaling properties of these family of several adapter proteins are required for full leukemic members. To gain insight into CRKL function, CRKL gene dis- potential.80 ruption may be useful. Finally, the role of CRKL in leukemic transformation, especially CML, is beginning to be defined and needs to be explored further. CRKL links BCR/ABL to the focal adhesion proteins paxillin and CAS Acknowledgements BCR/ABL induces global abnormalities of cytoskeletal func- tion,81 alters adhesion properties to extracellular matrix ´ This work was supported by Jose Carreras International Leuke- components such as fibronectin,82,83 and alters cell motility as mia Foundation fellowship FIJC-95/INT (MS) and NIH grant reflected by membrane ruffling, filipodia/lamellipodia, and CA75348 (RS). We would like to thank Dr James D Griffin for .84 Unlike c-ABL, BCR/ABL is primarily localized his support and critical comments on the manuscript. Also, to the actin cytoskeleton85 and is associated with focal we greatly appreciate the constructive criticism on the manu- adhesion proteins.81 BCR/ABL-transformed cells have an alt- script by Dr Vicky A Boussiotis. ered F-actin structure and have tyrosine phosphorylation of focal adhesion proteins such as tensin, , , RAFTK, p125FAK, and paxillin.28,33,86 CRKL is the key signaling mol- ecule which links BCR/ABL to the focal adhesion protein pax- References illin.33 Paxillin is a 68 kDa multi-functional protein with three YXXP motifs at the N-terminus and four LIM domains at the 1 ten Hoeve J, Morris C, Heisterkamp N, Groffen J. Isolation and C-terminus.87 By mutational analysis, Y31XXP and Y118XXP in chromosomal localization of CRKL, a human Crk-like gene. 33 Oncogene 1993; 8: 2469–2474. paxillin have been shown to bind to the CRKL SH2 domain 2 Oda T, Heaney C, Hagopian JR, Okuda K, Griffin JD, Druker BJ. 32,88 and to the CRK SH2 domain. 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