Activation of Hematopoietic Progenitor Kinase 1 Involves Relocation, Autophosphorylation, and Transphosphorylation by Protein Kinase D1

Abstract

Adaptive immune signaling can be coupled to stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) and NF-κB activation by the hematopoietic progenitor kinase 1 (HPK1), a mammalian hematopoiesis-specific Ste20 kinase. To gain insight into the regulation of leukocyte signal transduction, we investigated the molecular details of HPK1 activation. Here we demonstrate the capacity of the Src family kinase Lck and the SLP-76 family adaptor protein Clnk (cytokine-dependent hematopoietic cell linker) to induce HPK1 tyrosine phosphorylation and relocation to the plasma membrane, which in lymphocytes results in recruitment of HPK1 to the contact site of antigen-presenting cell (APC)-T-cell conjugates. Relocation and clustering of HPK1 cause its enzymatic activation, which is accompanied by phosphorylation of regulatory sites in the HPK1 kinase activation loop. We show that full activation of HPK1 is dependent on autophosphorylation of threonine 165 and phosphorylation of serine 171, which is a target site for protein kinase D (PKD) in vitro. Upon T-cell receptor stimulation, PKD robustly augments HPK1 kinase activity in Jurkat T cells and enhances HPK1-driven SAPK/JNK and NF-κB activation; conversely, antisense down-regulation of PKD results in reduced HPK1 activity. Thus, activation of major lymphocyte signaling pathways via HPK1 involves (i) relocation, (ii) autophosphorylation, and (iii) transphosphorylation of HPK1 by PKD.

ACKNOWLEDGMENTS

We thank Wolfgang Müller for expert technical assistance and Martin Stehling and Klaus Hexel for help with cell sorting. We are indebted to Alf Hamann, Siegfried Weiβ, and Jane McGlade for the gift of murine T- and B-cell lines and Min Li-Weber for providing the AP-1 reporter construct. We thank Kristina Brauburger and Marcel Deckert for valuable discussions. We are grateful to Christian R. Frey and Jörg Kraus for critically reading the manuscript and Peter H. Krammer for stimulating discussions and valuable contributions. We thank Dietmar Vestweber for his continued interest and generous support. J.V.L. thanks Sandy Vandoninck for expert technical assistance.

Research in the J.V.L. lab was supported by the Association for International Cancer Research (AICR grant 02-252), the Belgian Federation against Cancer (SCIE2003-53), the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen (FWO-Vl), and the Belgian government (IUAP P5/12). Research in the F.K. lab was supported by the Max-Planck-Society.