**1. Introduction**

112 Advances in Hematopoietic Stem Cell Research

Stark, G.R., Kerr, I.M., Williams B.R, Silverman, RH. & Schreiber, R.D. (1998). How cells

Stellacci, E., Testa, U., Petrucci, E., Benedetti, E., Orsatti, R., Feccia, T., Stafsnes, M., Marziali,

Taniguchi, T., Ogasawara, K., Takaoka, A. & Tanaka N. (2001). IRF family of transcription factors as regulators of host defense. *Annu Rev Immunol* Vol.19, pp. 623-655. Taniguchi, T. & Takaoka, A. (2001). A weak signal for strong responses: interferon-/ revisited. *Nat. Rev. Mol. Cell Biol* Vol. 2, No.5, (May 2001), pp. 378-386. Vaughan, P., Aziz, F., van Wijnen, A., Wu, S., Harada, H., Taniguchi ,T., Soprano, K.J., Stein,

G. & Battistini, A. (2004). Interferon regulatory factor-2 drives megakaryocytic

J.L. & Stein, G.S. (1995). Activation of a cell-cycle-regulated histone gene by the oncogenic transcription factor IRF-2. *Nature* Vol.377, 6547, (Sep 1995), pp. 362-365. Xi, H., Eason, D., Ghosh, D., Dovhey, S., Wright, K. & Blanck, G. (1999). Co-occupancy of the

interferon regulatory element of the class II transactivator (CIITA) type IV promoter by interferon regulatory factors 1 and 2. *Oncogene* Vol.18, No.43, (Oct

respond to interferons. *Annu Rev Biochem* Vol. 67, pp. 227-264.

differentiation. *Biochem J* Vol. 377, Pt2, (Jan 2004), pp. 367-378.

1999), pp. 5889-5903.

Phosphorylation of tyrosine residues is an essential biochemical reaction in many higher eukaryotes. One of the most important and well-studied functions of tyrosine phosphorylation is to convey extracellular signals to the cytoplasm and ultimately to the nucleus in order to control various cell functions such as proliferation, differentiation, migration and survival.

The first tyrosine kinase (TK) was discovered as the tumor-inducing activity from Rous sarcoma virus, which is now known as v-Src (Rous, 1911). Later studies revealed that the oncogenic properties of v-Src was due to the loss of the regulatory mechanisms to control its kinase activity (Martin, 2001). These findings clearly highlight the critical importance of precise regulation of TK activities in order to avoid detrimental consequences to the homeostasis of the organisms.

Various mechanisms are employed to control TK activities. In the Src family non-receptor tyrosine kinase (non-RTK), phosphorylation status of the tyrosine residue in the C-terminal regulatory region alters intramolecular interactions and therefore serves as a way to modulate kinase activity. The kinases and the phosphatases involved in this regulatory mechanism are, in turn, themselves under additional layers of regulation, thus, creating an intricate network of signal mediators to fine-tune cellular responses (Sen & Johnson, 2011). Incidentally, activity of a typical receptor tyrosine kinase (RTK) is regulated by ligand binding; RTKs alter conformation upon ligand binding and dimerize, which leads to transphosphorylation of critical tyrosine residues in the activation loop of the neighboring kinase in the cytoplasmic domain (He & Hristova). This initiates a cascade of biochemical reactions that activates downstream signaling pathways.

Subcellular localization of TKs is another important determinant of their activity (Murphy et al., 2009). There are now abundant evidence indicating that TKs can generate different signals dependent on their intracellular locations. Therefore, molecules that regulate protein trafficking and localization constitute a critical component of signal regulatory mechanism.

Covalent attachment of small proteins such as ubiquitin and small ubiquitin-like modifier (SUMO) to target proteins serves as a signal for various biological processing, including

Regulation of Tyrosine Kinase Signaling by Cbl in Hematopoietic Stem Cells 115

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,

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

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 proline-

linker; RF, RING finger; Y, tyrosine; UBA, ubiquitin-associated.

rich region (Rivero-Lezcano et al., 1994; Fukazawa et al., 1995).

sequences.

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

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 hematopoietic homeostasis.

Here, we review functions of the Cbl family proteins and some of the candidate Cbl targets in the HSC compartment and discuss potential mechanisms of their regulation.
