**2. The Cbl family proteins**

The Cbl family proteins are evolutionarily conserved signal regulators present through *C. elegans* to human (Figure 1). In mammals, this family includes Cbl (also known as c-Cbl, encoded by the *CBL* gene in human), Cbl-b (*CBLB* gene in human) and Cbl-c (also know as Cbl-3 or Cbl-SL, *CBLC* gene in human). Cbl was originally identified as a cellular homolog of a viral oncogene *v-Cbl* which caused leukemia and lymphoma in mice (Langdon et al., 1989). Cbl's involvement in signal transduction was suggested because it became prominently tyrosine-phosphorylated upon stimulation through various cell surface receptors (Donovan et al., 1994; Galisteo et al., 1995). But it was not until genetic studies in *C. elegans* identified the *sli-1* gene product as a Cbl homolog that Cbl was established as a negative regulator of RTK signaling.

#### **2.1 Structure and biochemical functions of the Cbl family proteins**

All Cbl family proteins share a high degree of homology in their N-terminal regions. These include the tyrosine kinase binding (TKB) domain, the RF domain and the short intervening linker region. X-ray crystallography studies revealed that the TKB domain comprised a fourhelix bundle (4H), a calcium-binding EF hand and a variant Src homology region 2 (SH2) domain (Meng et al., 1999). The TKB domain mediates specific binding to cognate phosphotyrosine-containing motifs in activated TKs and select non-TK signal mediators (Lupher et al., 1996). The RF domain and the linker region together bind to E2 ubiquitinconjugating enzymes and both of these motifs are essential for the E3 ubiquitin ligase activity of the Cbl family proteins (Joazeiro et al., 1999; Levkowitz et al., 1999; Yokouchi et al., 1999; Zheng et al., 2000).

alteration of its localization and promotion of degradation (Schulman & Harper, 2009; van Wijk & Timmers, 2010). This reaction is mediated by a series of biochemical reactions involving the E1 or activating enzyme, the E2 or conjugating enzyme and the E3 ligase. Human genome encodes for two E1s, thirty E2 and over one thousand E3s for the ubiquitin system. This pathway architecture immediately implies that the substrate specificity of the

The Casitas B-lineage lymphoma (Cbl) family proteins are RING finger (RF)-containing multi-domain adaptors that function as E3 ubiquitin ligase primarily towards activated TKs (Thien & Langdon, 2001; Duan et al., 2004; Schmidt & Dikic, 2005). Using geneticallyengineered mouse models, we and others showed that loss of Cbl, either singly or in combination with another family member Cbl-b, led to the enlargement of the hematopoietic stem cell (HSC) compartment (Naramura et al., 2011a). Additionally, mutations in the *CBL* gene have been identified in a small but significant number of hematological malignancies in human, and experimental evidence proved the oncogenicity of mutant *CBL* products (Naramura et al., 2011b). All together, these observations strongly support that the Cbl family proteins are critical regulators of

Here, we review functions of the Cbl family proteins and some of the candidate Cbl targets

The Cbl family proteins are evolutionarily conserved signal regulators present through *C. elegans* to human (Figure 1). In mammals, this family includes Cbl (also known as c-Cbl, encoded by the *CBL* gene in human), Cbl-b (*CBLB* gene in human) and Cbl-c (also know as Cbl-3 or Cbl-SL, *CBLC* gene in human). Cbl was originally identified as a cellular homolog of a viral oncogene *v-Cbl* which caused leukemia and lymphoma in mice (Langdon et al., 1989). Cbl's involvement in signal transduction was suggested because it became prominently tyrosine-phosphorylated upon stimulation through various cell surface receptors (Donovan et al., 1994; Galisteo et al., 1995). But it was not until genetic studies in *C. elegans* identified the *sli-1* gene product as a Cbl homolog that Cbl was established as a

All Cbl family proteins share a high degree of homology in their N-terminal regions. These include the tyrosine kinase binding (TKB) domain, the RF domain and the short intervening linker region. X-ray crystallography studies revealed that the TKB domain comprised a fourhelix bundle (4H), a calcium-binding EF hand and a variant Src homology region 2 (SH2) domain (Meng et al., 1999). The TKB domain mediates specific binding to cognate phosphotyrosine-containing motifs in activated TKs and select non-TK signal mediators (Lupher et al., 1996). The RF domain and the linker region together bind to E2 ubiquitinconjugating enzymes and both of these motifs are essential for the E3 ubiquitin ligase activity of the Cbl family proteins (Joazeiro et al., 1999; Levkowitz et al., 1999; Yokouchi et

in the HSC compartment and discuss potential mechanisms of their regulation.

**2.1 Structure and biochemical functions of the Cbl family proteins** 

ubiquitin system must be achieved largely at the level of E3s.

hematopoietic homeostasis.

**2. The Cbl family proteins** 

negative regulator of RTK signaling.

al., 1999; Zheng et al., 2000).

Fig. 1. Structure of the Cbl family proteins. The original oncogenic form of Cbl (v-Cbl), the three mammalian Cbl family proteins (Cbl, Cbl-b and Cbl-c), the short and long forms of *Drosophila* Cbl (D-CblS and D-CblL) and the *C. elegans* homolog (SLI-1) are shown. TKB, tyrosine kinase binding; 4H, four-helix bundle; EF, EF hand; SH2, Src homology region 2; L, linker; RF, RING finger; Y, tyrosine; UBA, ubiquitin-associated.

Band and colleagues originally described that the Cbl TKB domain specifically recognized the phosphotyrosine-containing motif D(N/D)XpY, which was later refined as (N/D)XpY(S/T)XXP, found in several TKs such as ZAP70, epidermal growth factor receptor (EGFR), and Src (Lupher et al., 1997). Additional binding motifs, RA(V/I)XNQpY(S/T) and DpYR, were proposed in the adaptor protein APS (Hu & Hubbard, 2005) and the RTK c-Met (also known as hepatocyte growth factor receptor; Peschard et al., 2004), respectively. A recent comprehensive structural study showed that phosphopeptides with diverse sequences bound TKB at the same site, albeit in two different orientations (Ng et al., 2008). These studies collectively revealed the unique binding strategy for the specialized and biologically vital function of the Cbl family proteins and provided means to identify potential Cbl targets based on the amino acid sequences.

The C-terminal half of the Cbl family proteins are more divergent. A proline-rich region follows the RF domain in all mammalian Cbl family proteins, but this domain is more prominent in Cbl and Cbl-b than in Cbl-c. Biochemical studies have demonstrated that Cbl interacted with SH3-domain containing proteins such as Grb2 and Nck through the prolinerich region (Rivero-Lezcano et al., 1994; Fukazawa et al., 1995).

Regulation of Tyrosine Kinase Signaling by Cbl in Hematopoietic Stem Cells 117

outwardly normal and had a normal lifespan. When both Cbl and Cbl-b are deleted in the HSC, however, mice succumbed to aggressive myeloproliferative disease-like leukemia

> (Murphy et al., 1998; Naramura et al., 1998; Thien et al., 1999; Molero et al., 2004; El Chami et al., 2005; Rathinam et al., 2008)

(Bachmaier et al., 2000; Chiang et al., 2000; Krawczyk et al., 2000; Chiang et al., 2007; Loeser et al., 2007; Bachmaier et

al., 2007)

within two to three months after birth (Naramura et al., 2010).

Altered T cell antigen receptor expression Increased tyrosine phosphorylation

Co-stiumlation-independent activation of

Predisposition to autoimmune diseases and

Table 1. Phenotypes of mice deficient in the Cbl family members

Resistance to spontaneous and transplanted tumors

*Cblc* No apparent phenotypes (Griffiths et al., 2003)

Because of the involvement of various RTKs in cancer, it has long been speculated that the Cbl family proteins may play critical roles in the initiation and/or progression of cancer. Oncogenic mutations in RTKs that abrogate interaction with Cbl have been reported (Peschard & Park, 2003), but the direct evidence supporting Cbl's roles in cancer was not

The vast majority of *CBL* mutations reported so far are associated with myeloid disorders. Although the first human *CBL* mutations were described in acute myeloid leukemia (AML) samples (Sargin et al., 2007; Caligiuri et al., 2007; Abbas et al., 2008), later studies documented a significant number of cases in myelodysplastic syndromes-myeloproliferative neoplasms (MDS/MPN), a heterogeneous group of myeloid disorders including the chronic myelomonocytic leukemia (CMML), atypical chronic myeloid leukemia (aCML) and juvenile myelomonocytic leukemia (JMML) (Dunbar et al., 2008; Reindl et al., 2009; Grand et al., 2009; Loh et al., 2009; Sanada et al., 2009; Makishima et al., 2009; Muramatsu et al., 2010; Fernandes et al., 2010; Niemeyer et al., 2010). The association of *CBL* mutations with JMML is particularly thought-provoking because the pathogenesis of this rare pediatric hematological malignancy is closely associated with the activation of the Ras-MAPK signaling pathway (Loh, 2011). Among JMML patients, the activating mutations of *PTPN11*, *NRAS* and *KRAS*, and the loss of *NF1*, a gene encoding for a Ras GTPase-activator account for approximately 75 % of the total cases. Roughly half of the remainder of the cases are now attributed to *CBL* mutations. While *in vitro* experimental data indicate that the loss of the Cbl family proteins lead to prolonged Erk activation, it was never formally demonstrated

Enhanced thymic selection

Decreased fertility Altered metabolism

peripheral T cells

inflammatory injury

**2.3 Cbl and hematological malignancies** 

whether Cbl can regulate Ras activity directly.

established until 2007.

*Cbl* 

*Cblb* 

Gene Phenotype Reference

Splenomegaly and extramedullary hematopoiesis

In addition to being a TK regulator, Cbl itself is subject to tyrosine phosphorylation. Phosphorylation at tyrosine residues 700, 731 and 774 have been extensively characterized; residues 700 and 774 provide docking sites for the SH2 domain-containing adaptor protein CrkL (Andoniou et al., 1996). Tyrosine 700 also mediates an interaction with the guanine nucleotide exchange factor Vav (Marengère et al., 1997). Tyrosine 731 provides a docking site of the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K) (Hunter et al., 1999). Based on sequence homology and experimental data, tyrosine residues in the Cterminal domain of Cbl-b are thought to share many of the same functions as those in Cbl. Cbl-c does not possess comparable tyrosines.

The C-termini of Cbl and Cbl-b, but not Cbl-c, contain a conserved domain known as a ubiquitin-associated (UBA) domain, which is present in a variety of proteins involved in ubiquitin-mediated processes. Structural studies indicate that this domain is capable of binding ubiquitin and involved in dimerization (Kozlov et al., 2007; Peschard et al., 2007).

### **2.2 Insights from genetic models**

The first critical clue into Cbl's functions came from genetic studies in *C. elegans* (Yoon et al., 1995). The vulval development in *C. elegans* is regulated by signals through the EGFR pathway. A reduction-of-function mutation in *let-23* (encodes the EGFR homolog) leads to death of most worms, and the vulval development is incomplete in surviving worms. However, when loss-of-function mutations in *sli-1* were introduced to this genetic background, worms survived and vulval development was restored. The sequence analysis of the *sli-1* gene revealed that it encoded a protein with a high similarity to Cbl, thus establishing Cbl as a negative regulator of the EGFR pathway.

Genetic studies in gene targeted mouse models provided further insights into the physiological roles of the Cbl family proteins in mammals. Cbl-deficient mice are viable, but they show recognizable changes in the hematopoietic, lymphoid, metabolic and reproductive systems. In contrast, effects of Cbl-b loss is mostly limited to the peripheral immune functions. Cbl-c expression appears to be restricted to the epithelial tissues, but no significant phenotypes were reported in mice deficient in Cbl-c (Table 1).

While mice deficient in either one of the Cbl family members are viable, simultaneous loss of Cbl and Cbl-b is not compatible with the survival of the organism and double-deficient mice do not survive beyond embryonic day 10 (Naramura et al., 2002). This indicates that Cbl and Cbl-b play redundant and overlapping functions in critical organ systems during fetal development. Using the Cre-loxP-mediated conditional gene deletion approach, effects of Cbl, Cbl-b loss have been analyzed in the T, B and HSC compartments (Naramura et al., 2002; Huang et al., 2006; Kitaura et al., 2007; Naramura et al., 2010). These studies demonstrated that, in the adaptive immune system, the Cbl family proteins are required to establish appropriate threshold for selection of T and B cells, and disruption of this process leads to autoimmune-like phenotypes in mice.

In the hematopoietic compartment, Cbl-deficiency leads to moderate splenomegaly and enhanced extramedullary hematopoiesis (Murphy et al., 1998). In the bone marrow, the lineage-negative, Sca-1-positive, c-Kit-positive (LSK) compartment, which is highly enriched for HSCs, is enlarged and Cbl-deficient HSCs showed enhanced capacity to reconstitute myeloabrated recipient's hematopoietic system (Rathinam et al., 2008). However, mice were

In addition to being a TK regulator, Cbl itself is subject to tyrosine phosphorylation. Phosphorylation at tyrosine residues 700, 731 and 774 have been extensively characterized; residues 700 and 774 provide docking sites for the SH2 domain-containing adaptor protein CrkL (Andoniou et al., 1996). Tyrosine 700 also mediates an interaction with the guanine nucleotide exchange factor Vav (Marengère et al., 1997). Tyrosine 731 provides a docking site of the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K) (Hunter et al., 1999). Based on sequence homology and experimental data, tyrosine residues in the Cterminal domain of Cbl-b are thought to share many of the same functions as those in Cbl.

The C-termini of Cbl and Cbl-b, but not Cbl-c, contain a conserved domain known as a ubiquitin-associated (UBA) domain, which is present in a variety of proteins involved in ubiquitin-mediated processes. Structural studies indicate that this domain is capable of binding ubiquitin and involved in dimerization (Kozlov et al., 2007; Peschard et al., 2007).

The first critical clue into Cbl's functions came from genetic studies in *C. elegans* (Yoon et al., 1995). The vulval development in *C. elegans* is regulated by signals through the EGFR pathway. A reduction-of-function mutation in *let-23* (encodes the EGFR homolog) leads to death of most worms, and the vulval development is incomplete in surviving worms. However, when loss-of-function mutations in *sli-1* were introduced to this genetic background, worms survived and vulval development was restored. The sequence analysis of the *sli-1* gene revealed that it encoded a protein with a high similarity to Cbl, thus

Genetic studies in gene targeted mouse models provided further insights into the physiological roles of the Cbl family proteins in mammals. Cbl-deficient mice are viable, but they show recognizable changes in the hematopoietic, lymphoid, metabolic and reproductive systems. In contrast, effects of Cbl-b loss is mostly limited to the peripheral immune functions. Cbl-c expression appears to be restricted to the epithelial tissues, but no

While mice deficient in either one of the Cbl family members are viable, simultaneous loss of Cbl and Cbl-b is not compatible with the survival of the organism and double-deficient mice do not survive beyond embryonic day 10 (Naramura et al., 2002). This indicates that Cbl and Cbl-b play redundant and overlapping functions in critical organ systems during fetal development. Using the Cre-loxP-mediated conditional gene deletion approach, effects of Cbl, Cbl-b loss have been analyzed in the T, B and HSC compartments (Naramura et al., 2002; Huang et al., 2006; Kitaura et al., 2007; Naramura et al., 2010). These studies demonstrated that, in the adaptive immune system, the Cbl family proteins are required to establish appropriate threshold for selection of T and B cells, and disruption of this process

In the hematopoietic compartment, Cbl-deficiency leads to moderate splenomegaly and enhanced extramedullary hematopoiesis (Murphy et al., 1998). In the bone marrow, the lineage-negative, Sca-1-positive, c-Kit-positive (LSK) compartment, which is highly enriched for HSCs, is enlarged and Cbl-deficient HSCs showed enhanced capacity to reconstitute myeloabrated recipient's hematopoietic system (Rathinam et al., 2008). However, mice were

Cbl-c does not possess comparable tyrosines.

establishing Cbl as a negative regulator of the EGFR pathway.

leads to autoimmune-like phenotypes in mice.

significant phenotypes were reported in mice deficient in Cbl-c (Table 1).

**2.2 Insights from genetic models** 

outwardly normal and had a normal lifespan. When both Cbl and Cbl-b are deleted in the HSC, however, mice succumbed to aggressive myeloproliferative disease-like leukemia within two to three months after birth (Naramura et al., 2010).


Table 1. Phenotypes of mice deficient in the Cbl family members

#### **2.3 Cbl and hematological malignancies**

Because of the involvement of various RTKs in cancer, it has long been speculated that the Cbl family proteins may play critical roles in the initiation and/or progression of cancer. Oncogenic mutations in RTKs that abrogate interaction with Cbl have been reported (Peschard & Park, 2003), but the direct evidence supporting Cbl's roles in cancer was not established until 2007.

The vast majority of *CBL* mutations reported so far are associated with myeloid disorders. Although the first human *CBL* mutations were described in acute myeloid leukemia (AML) samples (Sargin et al., 2007; Caligiuri et al., 2007; Abbas et al., 2008), later studies documented a significant number of cases in myelodysplastic syndromes-myeloproliferative neoplasms (MDS/MPN), a heterogeneous group of myeloid disorders including the chronic myelomonocytic leukemia (CMML), atypical chronic myeloid leukemia (aCML) and juvenile myelomonocytic leukemia (JMML) (Dunbar et al., 2008; Reindl et al., 2009; Grand et al., 2009; Loh et al., 2009; Sanada et al., 2009; Makishima et al., 2009; Muramatsu et al., 2010; Fernandes et al., 2010; Niemeyer et al., 2010). The association of *CBL* mutations with JMML is particularly thought-provoking because the pathogenesis of this rare pediatric hematological malignancy is closely associated with the activation of the Ras-MAPK signaling pathway (Loh, 2011). Among JMML patients, the activating mutations of *PTPN11*, *NRAS* and *KRAS*, and the loss of *NF1*, a gene encoding for a Ras GTPase-activator account for approximately 75 % of the total cases. Roughly half of the remainder of the cases are now attributed to *CBL* mutations. While *in vitro* experimental data indicate that the loss of the Cbl family proteins lead to prolonged Erk activation, it was never formally demonstrated whether Cbl can regulate Ras activity directly.

Regulation of Tyrosine Kinase Signaling by Cbl in Hematopoietic Stem Cells 119

T cell antigen receptor complex (Donovan et al., 1994; Meisner et al., 1995;

B cell antigen receptor complex (Cory et al., 1995; Tezuka et al., 1996;

Fc receptor (Matsuo et al., 1996; Suzuki et al., 1997)

Epidermal growth factor receptor (Galisteo et al., 1995)

 Fibroblast growth factor receptor (Wong et al., 2002) Colony stimulating factor 1 receptor (Wang et al., 1996) Met (Fixman et al., 1997; Garcia-Guzman et al., 2000)

Platelet-derived growth factor receptor (Bonita et al., 1997)

Erythropoietin receptor (Odai et al., 1995; Barber et al., 1997)

Integrins (Ojaniemi et al., 1997; Manié et al., 1997; Meng & Lowell, 1998)

The mouse dominant spotting mutation at the *W* locus was first described in the early 1900s (Durham, 1908). Mutations at this locus were studied extensively not only because they produced visible coat color changes, but also because mutant mice showed defects in hematopoiesis, mast cell development and gametogenesis (Russell, 1979). However, it was not until 1988 that the gene product at the *W* locus was found to encode for the cellular homolog of the *kit* oncogene which had been molecularly identified a few years earlier

Kit is a type III RTK that shares structural similarities with platelet-derived growth factor receptors (PDGFRs) and , Flt3 (also known as Flk-2, discussed below) and CSF1R. They

Antigen and other immunological receptors

Panchamoorthy et al., 1996) Fc receptor (Marcilla et al., 1995)

Insulin receptor (Ribon & Saltiel, 1997)

Flt3 (Lavagna-Sévenier et al., 1998)

TrkB (McCarty & Feinstein, 1999)

 Interleukin 2 receptor (Gesbert et al., 1998) Interleukin 3 receptor (Barber et al., 1997) Interleukin 4 receptor (Ueno et al., 1998)

 Mpl (Sasaki et al., 1995; Brizzi et al., 1996) GM-CSF receptor (Odai et al., 1995) Prolactin receptor (Hunter et al., 1997)

Table 2. Partial list of potential upstream receptors for Cbl

(Besmer et al., 1986; Chabot et al., 1988; Geissler et al., 1988).

Chemokine receptors (Chernock et al., 2001)

Tie2 (Wehrle et al., 2009)

Cytokine receptors

**3. Kit** 

Kit (Wisniewski et al., 1996; Brizzi et al., 1996)

Fukazawa et al., 1995)

RTKs

It is of note that most *CBL* mutations are either point mutation or internal deletion involving the linker and/or the RF regions rather than complete deletion at the *CBL* locus. As expected from domain-function analysis results, these mutant Cbl proteins lack E3 ubiquitin ligase activity. Interestingly, in patient samples with *CBL* mutations, the wildtype allele is often lost and replaced with the mutant allele by acquired uniparental isodisomy (aUPD). The *CBLB*, *CBLC* alleles are usually unaffected in these patients although mutations in these genes have been reported (Makishima et al., 2009; Makishima et al., 2011). All together, these clinical observations suggest that the presence of one wildtype copy of *CBL* is generally sufficient to maintain the functions of Cbl in the presence of normal Cbl-b and Cbl-c. These findings are consistent with the data in mice expressing a RF-mutant Cbl from the endogenous promoter on a *Cblb*, *Cblc* wild-type background (Thien et al., 2005; Rathinam et al., 2010); homozygous mutant mice are perinatally lethal, but hemizygous mutants over the wild-type *Cbl* allele develop normally. However, when the hemizygous mutant is expressed over the Cbl-null background, mice develop myeloproliferative disease-like leukemia within a year. This is a striking contrast when compared to the rapid progression and fatality of the HSC-specific Cbl, Cbl-b doubledeficient mice (Naramura et al., 2010). These differences may reflect that the RF mutant and patient-derived oncogenic mutant Cbl proteins function as gain-of-function mutants rather than as dominant-negative inhibitors of Cbl-b (Cbl-c expression is minimal in the hematopoietic system). While these mutants lack E3 ubiquitin ligase activity and thus defective in promoting target degradation, they possess intact TKB and C-terminal protein-protein interaction motifs, which may enable them to form aberrant but stable multi-protein super-signaling complexes and activate unconventional signaling pathways.

#### **2.4 Potential Cbl targets in the hematopoietic system**

What, then, are the target of Cbl-dependent regulation in the HSC compartment? Because Cbl becomes phosphorylated upon stimulation with various cell surface receptors, it is conceivable that Cbl is involved in the regulation of signal transduction downstream of such pathways (Table 2). Among this diverse group of cell surface receptors, Kit and Flt3 are of particular interest because both of them are RTKs expressed in HSC and known to perform critical functions in the HSC compartment (Masson & Rönnstrand, 2009). Colony stimulating factor 1 receptor (CSF1R) is known to interact with Cbl (Lee et al., 1999), but it is expressed primarily in more differentiated myeloid/phagocytic cells than in HSCs. Endothelial-specific receptor tyrosine kinase (Tek, also known as Tie2) is another RTK expressed in the HSC compartment (Arai et al., 2004) and therefore may interact with Cbl. Thrombopoietin (TPO) is indispensable for the maintenance of HSC quiescence (Yoshihara et al., 2007; Qian et al., 2007). Although its receptor (TPO-R, also known as Mpl or c-Mpl) is not an RTK, stimulation with TPO induce phosphorylation of Cbl (Sasaki et al., 1995), Mpl have been shown to be ubiquitinylated (Saur et al., 2010) and Cbl loss alters the signal transduction downstream of TPO (Rathinam et al., 2008; Naramura et al., 2010). Therefore, Mpl may interact with Cbl indirectly. Other potential (direct as well as indirect) Cbl targets in the HSC compartment include the chemokine and integrin pathways.

In following sections, I will discuss how these pathways may be regulated by the Cbl family proteins in the HSCs.

Antigen and other immunological receptors


#### RTKs

118 Advances in Hematopoietic Stem Cell Research

It is of note that most *CBL* mutations are either point mutation or internal deletion involving the linker and/or the RF regions rather than complete deletion at the *CBL* locus. As expected from domain-function analysis results, these mutant Cbl proteins lack E3 ubiquitin ligase activity. Interestingly, in patient samples with *CBL* mutations, the wildtype allele is often lost and replaced with the mutant allele by acquired uniparental isodisomy (aUPD). The *CBLB*, *CBLC* alleles are usually unaffected in these patients although mutations in these genes have been reported (Makishima et al., 2009; Makishima et al., 2011). All together, these clinical observations suggest that the presence of one wildtype copy of *CBL* is generally sufficient to maintain the functions of Cbl in the presence of normal Cbl-b and Cbl-c. These findings are consistent with the data in mice expressing a RF-mutant Cbl from the endogenous promoter on a *Cblb*, *Cblc* wild-type background (Thien et al., 2005; Rathinam et al., 2010); homozygous mutant mice are perinatally lethal, but hemizygous mutants over the wild-type *Cbl* allele develop normally. However, when the hemizygous mutant is expressed over the Cbl-null background, mice develop myeloproliferative disease-like leukemia within a year. This is a striking contrast when compared to the rapid progression and fatality of the HSC-specific Cbl, Cbl-b doubledeficient mice (Naramura et al., 2010). These differences may reflect that the RF mutant and patient-derived oncogenic mutant Cbl proteins function as gain-of-function mutants rather than as dominant-negative inhibitors of Cbl-b (Cbl-c expression is minimal in the hematopoietic system). While these mutants lack E3 ubiquitin ligase activity and thus defective in promoting target degradation, they possess intact TKB and C-terminal protein-protein interaction motifs, which may enable them to form aberrant but stable multi-protein super-signaling complexes and activate unconventional signaling pathways.

What, then, are the target of Cbl-dependent regulation in the HSC compartment? Because Cbl becomes phosphorylated upon stimulation with various cell surface receptors, it is conceivable that Cbl is involved in the regulation of signal transduction downstream of such pathways (Table 2). Among this diverse group of cell surface receptors, Kit and Flt3 are of particular interest because both of them are RTKs expressed in HSC and known to perform critical functions in the HSC compartment (Masson & Rönnstrand, 2009). Colony stimulating factor 1 receptor (CSF1R) is known to interact with Cbl (Lee et al., 1999), but it is expressed primarily in more differentiated myeloid/phagocytic cells than in HSCs. Endothelial-specific receptor tyrosine kinase (Tek, also known as Tie2) is another RTK expressed in the HSC compartment (Arai et al., 2004) and therefore may interact with Cbl. Thrombopoietin (TPO) is indispensable for the maintenance of HSC quiescence (Yoshihara et al., 2007; Qian et al., 2007). Although its receptor (TPO-R, also known as Mpl or c-Mpl) is not an RTK, stimulation with TPO induce phosphorylation of Cbl (Sasaki et al., 1995), Mpl have been shown to be ubiquitinylated (Saur et al., 2010) and Cbl loss alters the signal transduction downstream of TPO (Rathinam et al., 2008; Naramura et al., 2010). Therefore, Mpl may interact with Cbl indirectly. Other potential (direct as well as indirect) Cbl targets in the HSC compartment include the chemokine and integrin

In following sections, I will discuss how these pathways may be regulated by the Cbl family

**2.4 Potential Cbl targets in the hematopoietic system** 

pathways.

proteins in the HSCs.


Cytokine receptors


Chemokine receptors (Chernock et al., 2001)

#### Integrins (Ojaniemi et al., 1997; Manié et al., 1997; Meng & Lowell, 1998)

Table 2. Partial list of potential upstream receptors for Cbl
