Sansana Sawasdikosol

Structure of the amino-terminal domain of Cbl complexed to its binding site on ZAP-70 kinase

Cbl is an adaptor protein that functions as a negative regulator of many signalling pathways that start from receptors at the cell surface. The evolutionarily conserved amino-terminal region of Cbl (Cbl-N) binds to phosphorylated tyrosine residues and has cell-transforming activity. Point mutations in Cbl that disrupt its recognition of phosphotyrosine also interfere with its negative regulatory function and, in the case of v-cbl, with its oncogenic potential. In T cells, Cbl-N binds to the tyrosine-phosphorylated inhibitory site of the protein tyrosine kinase ZAP-70. Here we describe the crystal structure of Cbl-N, both alone and in complex with a phosphopeptide that represents its binding site in ZAP-70. The structures show that Cbl-N is composed of three interacting domains: a four-helix bundle (4H), an EF-hand calcium-binding domain, and a divergent SH2 domain that was not recognizable from the amino-acid sequence of the protein. The calcium-bound EF hand wedges between the 4H and SH2 domains and roughly determines their relative orientation. In the ligand-occupied structure, the 4H domain packs against the SH2 domain and completes its phosphotyrosine-recognition pocket. Disruption of this binding to ZAP-70 as a result of structure-based mutations in the 4H, EF-hand and SH2 domains confirms that the three domains together form an integrated phosphoprotein-recognition module.

The Small GTP-Binding Protein Rho Potentiates AP-1 Transcription in T Cells

ABSTRACT The Rho family of small GTP-binding proteins is involved in the regulation of cytoskeletal structure, gene transcription, specific cell fate development, and transformation. We demonstrate in this report that overexpression of an activated form of Rho enhances AP-1 activity in Jurkat T cells in the presence of phorbol myristate acetate (PMA), but activated Rho (V14Rho) has little or no effect on NFAT, Oct-1, and NF-κB enhancer element activities under similar conditions. Overexpression of a V14Rho construct incapable of membrane localization (CAAX deleted) abolishes PMA-induced AP-1 transcriptional activation. The effect of Rho on AP-1 is independent of the mitogen-activated protein kinase pathway, as a dominant-negative MEK and a MEK inhibitor (PD98059) did not affect Rho-induced AP-1 activity. V14Rho binds strongly to protein kinase Cα (PKCα) in vivo; however, deletion of the CAAX site on V14Rho severely diminished this association. Evidence for a role for PKCα as an effector of Rho was obtained by the observation that coexpression of the N-terminal domain of PKCα blocked the effects of activated Rho plus PMA on AP-1 transcriptional activity. These data suggest that Rho potentiates AP-1 transcription during T-cell activation.

A Peptide Library Approach Identifies a Specific Inhibitor for the ZAP-70 Protein Tyrosine Kinase

We utilized a novel peptide library approach to identify specific inhibitors of ZAP-70, a protein Tyr kinase involved in T cell activation. By screening more than 6 billion peptides oriented by a common Tyr residue for their ability to bind to ZAP-70, we determined a consensus optimal peptide. A Phe-for-Tyr substituted version of the peptide inhibited ZAP-70 protein Tyr kinase activity by competing with protein substrates (K(I) of 2 microM). The related protein Tyr kinases, Lck and Syk, were not significantly inhibited by the peptide. When introduced into intact T cells, the peptide blocked signaling downstream of ZAP-70, including ZAP-70-dependent gene induction, without affecting upstream Tyr phosphorylation. Thus, screening Tyr-oriented peptide libraries can identify selective peptide inhibitors of protein Tyr kinases.

Prostaglandin E2 Activates HPK1 Kinase Activity via a PKA-dependent Pathway

Hematopoietic progenitor kinase 1 (HPK1) is a hematopoietic cell-restricted member of the Ste20 serine/threonine kinase super family. We recently reported that the immunosuppressive eicosanoid, prostaglandin E2 (PGE2), is capable of activating HPK1 in T cells. In this report, we demonstrate that unlike the TCR-induced activation of HPK1 kinase activity, the induction of HPK1 catalytic activity by PGE2 does not require the presence of phosphotyrosine-based signaling molecules such as Lck, ZAP-70, SLP-76, and Lat. Nor does the PGE2-induced HPK1 activation require the intermolecular interaction between its proline-rich regions and the SH3 domain-containing adaptor proteins, as required by the signaling from the TCR to HPK1. Instead, our study reveals that PGE2 signal to HPK1 via a 3′ -5 ′-cyclic adenosine monophosphate-regulated, PKA-dependent pathway. Consistent with this observation, changing the serine 171 residue that forms the optimal PKA phosphorylation site within the “activation loop” of HPK1 to alanine completely prevents this mutant from responding to PGE2-generated stimulation signals. Moreover, the inability of HPK1 to respond to PGE2 stimulation in PKA-deficient S49 cells further supports the importance of PKA in this signaling pathway. We speculate that this unique signaling pathway enables PGE2 signals to engage a proven negative regulator of TCR signal transduction pathway and uses it to inhibit T cell activation.

Hematopoietic progenitor kinase 1 is a critical component of prostaglandin E2-mediated suppression of the anti-tumor immune response

Lung cancer is the leading cause of cancer-related mortality in the world, resulting in over a million deaths each year. Non-small cell lung cancers (NSCLCs) are characterized by a poor immunogenic response, which may be the result of immunosuppressive factors such as prostaglandin E2 (PGE2) present in the tumor environment. The effect of PGE2 in the suppression of anti-tumor immunity and its promotion of tumor survival has been established for over three decades, but with limited mechanistic understanding. We have previously reported that PGE2 activates hematopoietic progenitor kinase 1 (HPK1), a hematopoietic-specific kinase known to negatively regulate T-cell receptor signaling. Here, we report that mice genetically lacking HPK1 resist the growth of PGE2-producing Lewis lung carcinoma (LLC). The presence of tumor-infiltrating lymphocytes (TILs) and T-cell transfer into T cell-deficient mice revealed that tumor rejection is T cell mediated. Further analysis demonstrated that this may be significantly due to the ability of HPK1−/− T cells to withstand PGE2-mediated suppression of T-cell proliferation, IL-2 production, and apoptosis. We conclude that PGE2 utilizes HPK1 to suppress T cell-mediated anti-tumor responses.

Hematopoietic Progenitor Kinase 1 Is a Negative Regulator of Dendritic Cell Activation

Hematopoietic progenitor kinase 1 (HPK1) is a hematopoietic cell-restricted member of the Ste20 kinases that acts as a negative regulator of T cell functions through the AP-1, NFAT, and NFκB pathways. Using HPK1-deficient (HPK1−/−) mice, we report in this study a novel role for HPK1 in dendritic cells (DCs). Specifically, we observed that matured HPK1−/− bone marrow-derived DCs (BMDCs) are superior to their wild-type (WT) counterpart in stimulating T cell proliferation in vivo and in vitro. Several characteristics of HPK1−/− BMDCs may account for this enhanced activity: Matured HPK1−/− BMDCs express higher levels of costimulatory molecules CD80, CD86, and I-Ab as well as produce more proinflammatory cytokines IL-12, IL-1β, TNF-α, and IL-6 than their WT littermates. The role of HPK1 as a proapoptotic molecule was assessed post activation with LPS, and results indicated that HPK1−/− BMDCs are significantly resistant to LPS-induced apoptosis. Our results led us to investigate the role of HPK1−/− BMDCs in tumor immunotherapy. Using a s.c. murine model of Lewis Lung Carcinoma, we found that HPK1−/− BMDCs eliminate established s.c. Lewis Lung Carcinoma more efficiently than their WT counterpart. Our data reveal a novel role for HPK1 as a negative regulator of DC functions, identifying its potential as a molecular target for DC-based immunotherapy against cancers.