BLNK binds active H-Ras to promote B cell receptor-mediated capping and ERK activation.

Cross-linked B cell receptor (BCR) aggregates on the cell surface, then assembles into the "cap" where Ras is co-localized, and transduces various intracellular signals including Ras-ERK activation. BCR signals induce proliferation, differentiation, or apoptosis of B cells depending on their maturational stage. The adaptor protein BLNK binds various signaling proteins and Igalpha, a signaling subunit of the BCR complex, and plays an important role in the BCR signal transduction. BLNK was shown to be required for activation of ERK, but not of Ras, after BCR cross-linking, raising a question how BLNK facilitates ERK activation. Here we demonstrate that BLNK binds the active form of H-Ras, and their binding is facilitated by BCR cross-linking. We have identified a 10-amino acid Ras-binding domain within BLNK that is necessary for restoration of BCR-mediated ERK activation in BLNK-deficient B cells and for anti-apoptotic signaling. The Ras-binding domain fused with a CD8alpha-Igalpha chimeric receptor could induce prolonged ERK phosphorylation, transcriptional activation of Elk1, as well as the capping of the receptor in BLNK-deficient B cells. These results indicate that BLNK recruits active H-Ras to the BCR complex, which is essential for sustained surface expression of BCR in the form of the cap and for the signal leading to functional ERK activation.

BLNK suppresses pre-B-cell leukemogenesis through inhibition of JAK3.

Pre-B-cell leukemia spontaneously develops in BLNK-deficient mice, and pre-B-cell acute lymphoblastic leukemia cells in children often lack BLNK protein expression, demonstrating that BLNK functions as a tumor suppressor. However, the mechanism by which BLNK suppresses pre-B-cell leukemia, as well as the identification of other genetic alterations that collaborate with BLNK deficiency to cause leukemogenesis, are still unknown. Here, we demonstrate that the JAK3/STAT5 signaling pathway is constitutively activated in pre-B leukemia cells derived from BLNK(-/-) mice, mostly due to autocrine production of IL-7. Inhibition of IL-7R signaling or JAK3/STAT5 activity resulted in the induction of p27(kip1) expression and cell-cycle arrest, accompanied by apoptosis in the leukemia cells. Transgene-derived constitutively active STAT5 (STAT5b-CA) strongly synergized with the loss of BLNK to initiate leukemia in vivo. In the leukemia cells, exogenously expressed BLNK inhibited autocrine JAK3/STAT5 signaling, resulting in p27(kip1) induction, cell-cycle arrest, and apoptosis. BLNK-inhibition of JAK3 was dependent on the binding of BLNK to JAK3. These data indicate that BLNK normally regulates IL-7-dependent proliferation and survival of pre-B cells through direct inhibition of JAK3. Thus, somatic loss of BLNK and concomitant mutations leading to constitutive activation of Jak/STAT5 pathway result in the generation of pre-B-cell leukemia.

Dok-1 and Dok-2 are negative regulators of T cell receptor signaling.

Interaction of the TCR complex with self- or foreign peptides is a central event in the immune responses. Upon TCR stimulation, a protein-tyrosine kinase (PTK), ZAP-70, is recruited to signaling units of the TCR complex, such as TCRzeta, to play an essential role in T cell activation. Here, we find that mice lacking adaptor proteins Dok-1 and Dok-2 show augmented responses to thymus-dependent, but not thymus-independent, antigens, and that their T cells show elevated responses to TCR stimulation, including the activation of ZAP-70 and subsequent proliferation and cytokine production. Furthermore, the forced expression of Dok-1 or Dok-2 in a CD3(+)CD4(+) T cell clone inhibited the activation of ZAP-70 upon TCR stimulation. Interestingly, the Dok-1 and Dok-2 COOH-terminal moieties bearing the src homology 2 target motifs were dispensable for this negative regulation, even though they are crucial for the known adaptor function of Dok-family proteins. Thus, by an as yet unidentified mechanism, Dok-1 and Dok-2 play an essential role in the negative regulation of TCR signaling. Consistently, all mice lacking these proteins exhibited elevated titers of antibodies to double-stranded DNA and developed lupus-like renal disease.