Abteilung Vaskuläre Zellbiologie
Leiter: Prof. Dr. Dietmar Vestweber

Abteilungsassoziierte Forschungsgruppe Dr. Friedemann Kiefer

Friedemann Kiefer Ph.D. E-mail: fkiefer at gwdg.de
phone ++49-251 / 70365 230
fax ++49-251 / 70365 299

Research subject
Signal transduction in endothelial cells and leukocytes

People in the laboratory
Ruben Böhmer (PhD student)
Brit Neuhaus (PhD student)
Irene Patzak (PhD student)
Barbara Waschk (technician)
Peter Schulze-Niehoff (technician)

Former lab members
Rüdiger Arnold Ph.D. (post-doctoral fellow) r.arnold at dkfz-heidelberg.de
Barbara Böck Ph.D. (post-doctoral fellow) b.boeck at dkfz-heidelberg.de

Signaling mechanisms in endothelial cells and leukocytes

All mammalian cells react in complex ways to a multitude of extracellular stimuli that can be as divers as growth and survival factors, mediators of inflammation, or toxic environmental conditions like UV light or irradiation. A large variety of receptors located at the cell membrane senses these extracellular signals and transmits them to the cell interior. Inside the cell information is conveyed by directly interconnected biochemical reactions called signal transduction pathways. Despite the huge number of receptors on their surface, cells employ only a limited number of evolutionarily highly conserved signaling pathways. To evoke the many distinct reactions specific to different cell types signaling pathways have to work in a combinatorial and graded fashion, using an istense network of positive and negative regulatory loops.

Ligand activation of cell surface receptors results in the rapid and reversible activation of intracellular enzymatic activities, most notable tyrosine phosphorylation. Kinase activity may be intrinsic to the receptor as in receptor tyrosine kinases (RTKs) or be exerted by recruited cellular non-receptor tyrosine kinases like members of the Src and Syk / ZAP-70 families. Protein kinases are equipped with the capacity to selectively recognize their target peptides for phosphorylation and employ phosphorylation to induce conformational changes in regulatory enzymes. However, a major breakthrough in the understanding of signaling specificity was the discovery of the SH2 domain and the realization that besides conformational enzymatic regulation tyrosine phosphorylation has the capacity to provide docking points for the selective recruitment of signaling intermediates.

It became clear over the past decade that activated cell surface receptors nucleate the formation of large complexes consisting of multiple adaptor proteins and various signaling enzymes. These membrane-proximal multi protein complexes are the key players in the regulated activation of the universal intracellular signaling chains. In particular the tissue specific expression pattern of cytoplasmic and trans-membraneous adaptor proteins dictates the formation of specific signaling complexes.

Important questions that remain to be addressed concern the dynamics of signal complex formation and their stability, possible competition between different complexes within the same cell and consequently the quality of the signals generated. The extent of activation of a particular pathway as well as the duration of the signal are likely having an important influence on the cellular response triggered.

Over the past years research in my laboratory was focussed on signaling mechanisms active in hematopoietic and endothelial cells. One emphasis lies in understanding the fine tuning of innate and adaptive immunity. In particular we have focussed on the function of the Ste20-related kinases germinal-center kinase (GCK) and hematopoietic progenitor kinase (HPK) in lymphocyte activation. HPK1 appears to fulfil an important switch function activating stress activated kinases (SAPKs) and NFκB transcription factor depending on the physiological state of the lymphocyte.

Our second research focus concerns the vascular system. Blood and lymphatic vessels arise early during embryonic development from common progenitors, which adopt distinct fates leading to the establishment of two separate vascular systems. Loss of the non-receptor tyrosine kinase Syk results in a loss of separation of blood and lymphatic vessels. We are studying the Syk knock out to gain insights into the mechanisms underlying the segregation of blood and lymphatic vessel and the processes that maintain their integrity and identity. The process appears to involve the interaction of different cell types, notably endothelial and mural cells as well as leukocytes. The scarcity of embryonic endothelium has largely precluded a biochemical analysis of this tissue; however, embryonic endothelium can be immortalized through the action of the polyomavirus middle T antigen (PymT). We have analyzed in detail the transforming complex formed by PymT in endothelioma cells and noted a striking similarity to the signaling complexes nucleated by genuine endothelial receptors.

Fine tuning of lymphocyte activation - Germinal Centre kinases as potential regulators of AICD, autoimmunity and allergy

Lymphocyte activation and adaptive immunity are highly controlled processes, deregulation of which can result in autoimmunity and allergy. Over the last decade tremendous progress has been made in the elucidation of the mechanisms of lymphocyte activation and the major classes of molecules governing this process. The mechanisms controlling the extent and fine tuning of immune responses are only beginning to emerge.

Several years ago, we have cloned the hematopoietic progenitor kinase 1 (HPK1) a mammalian serine/threonine kinase that bears homology to the Ste20 kinase of Saccharomyces cerevisiae. HPK1 is exclusively expressed in hematopoietic progenitor cells and mature leukocytes (Kiefer et al., 1996) and is activated by immunoreceptors (Liou et al., 2000). Activation necessitates formation of a membrane proximal complex entailing besides HPK1, adaptor proteins of the Grb2 and SLP-76 (SH2-domain containing leukocyte protein of 76 kDa) families (Liu et al., 2000), (Tsuji et al., 2001), (Yu et al., 2001). In this complex HPK1 is subject to serine, threonine and tyrosine phosphorylation. Transphosphorylation of tyrosine 376 after by Syk provides a docking site for the SH2 domain of SLP76 adaptor proteins. This phosphorylation event helps to localize HPK1 to the membrane. Using phosphopeptide mapping we were able to identify PKD1 as a HPK1-kinase and demonstrate that phosphorylation of serine 171 by PKD1 provides a priming phosphorylation for HPK1 autophosphorylation on threonine 165 (Arnold et al., 2005). Full activation of HPK1 necessitates the concerted progression through all phosphorylation events.

Once fully active, HPK1 is a selective and potent activator of the SAPK / JNK MAP-kinases as well as the NFκB transcriptions factors. In apoptotic cells, HPK1 is subject to caspase cleavage converting it to an inhibitor of NFκB activation, while its capacity to stimulate SAPKs is retained (Arnold et al., 2001). Studies using transgenic mice which express a dominant-negative version of HPK1 have provided solid evidence that HPK1 can sensitize primary T-cells to activation induced cell death (AICD) (Brenner et al., 2005). HPK1-deficient mice show signs of hyperreactivity when exposed to particular stimuli suggesting functions in the suppression of autoimmunity and allergic reactions (Patzak, Kiefer unpublished). By acting as a life / death switch and a negative modulator of lymphocyte activation HPK1 essentially contributes to specific aspects of immune homeostasis.

Development and maintenance of vascular integrity and mechanisms maintaining the identity of blood and lymphatic vessels.

Polyomavirus middle T-antigen allows the identification of signaling pathways active during endothelial differentiation.

Middle T antigen (PymT) is the principal oncogene of murine polyomavirus. It rapidly induces lethal endothelial tumors (hemangiomas) in embryonic and neonatal mice from which PymT-transformed endothelial cells (endothelioma cells) can be derived.

PymT, a membrane-associated scaffold, recruits and activates Src family tyrosine kinases. Once tyrosine phosphorylated, PymT binds proteins with phosphotyrosine binding (PTB) and SH2 domains such as the adaptor protein ShcA, phosphatidylinositol 3-kinase (PI 3-K) and phospholipase Cγ-1 (PLCγ-1). We found a PymT mutant unable to bind ShcA to be transformation compromised suggesting a function of ShcA in the control of endothelial proliferation. Subsequent detailed biochemical analysis demonstrated that in transformed endothelial cells PymT mimics activated receptor tyrosine kinases by establishing a large signaling platform at the plasma membrane that contains adaptor proteins such as ShcA, Grb2 and Gab2 (Ong et al., 2001). Interestingly only embryonic and newborn endothelium is susceptible to PymT-mediated transformation, mice infected at later age fail to develop hemangiomas. Apparently PymT precisely usurps a part of the cellular signaling machinery that is endogenously employed by growing endothelial cells, rendering this cell type exquisitely sensitive to PymT action.

As a tool PymT can be exploited to generate endothelioma cell lines from various knock out mouse strains. This allows the generation of tissue culture models for the functional and biochemical analysis of these genes in endothelial cells.

Maintenance and separation of blood and lymphatic endothelial cells - the non-receptor tyrosine kinase Syk as a major regulator of endothelial mobility.

Vascular integrity is a prerequisite for the function of all organ systems throughout life. A number of growth factors and cognate receptors most notably of the VEGF and angiopoietin family have been demonstrated to govern the formation of the vascular system. To date the mechanisms regulating blood vessel formation are far better understood then the mechanisms that determine lymphatic vessels and the regulatory networks which control maintenance of both systems and assure their function as separate identities are yet to be described in detail.

The cytoplasmic non-receptor tyrosine kinase Syk is a well characterized signal transducer in B-cells, granulocytes, macrophages and platelets. Syk mediates immunoreceptor and integrin triggered signals resulting in activation of the prototypic Erk, SAPK, PI-3 kinase and PLC-γ cascades. Knockout studies in mice demonstrated an essential function of Syk in the development of B- and γ/δ T-cells. Unexpectedly, Syk-deficient mice die in utero from lethal hemorrhages that are characterized by endothelial cell death and arterio-venous lymphatic shunting. We and others have shown that this break-down of vascular integrity is recapitulated in adult bone marrow chimeras reconstituted with Syk-deficient fetal liver cells (Kiefer et al., 1998). Also in transplanted adults the separation between blood and lymphatic vessels is lost and inappropriate shunting occurs, leading to chylus ascites, hemorrhaging and death. The underlying molecular mechanisms are elusive, however, platelets defects due to Syk-deficiency can be excluded to cause the lethal hemorrhages.

We hypothesised that the lymphatic dys-morphogenesis and bleeding disorder are related to possible novel functions of the non-receptor tyrosine kinase Syk in extracellular matrix signaling. Such functions may involve leukocytes, endothelial cells and likely interactions between both cell types. We found that vascular morphogenesis was impaired in developing Syk-deficient embryos, before the onset of lymphangiogenesis and that Syk is expressed in HUVEC cells and endothelioma cells derived from midgestation embryos. Endothelioma cells are a valuable model system for the bleeding affliction. Syk activity is modulated depending on extracellular matrix (ECM) components provided for the growth of endotheliomas and Syk activity results in significant changes in the mobility of endothelioma cells. Our current working hypothesis assumes that Syk modulates endothelial motility during embryogenesis and thereby contributes to the separation of vascular and lymphatic endothelial cells (Neuhaus, Kiefer unpublished).

Fig 1: The hematopoietic serine threonine kinase HPK1 is activated in response to immunoreceptor triggering. Enzymatic activation of HPK1 entails translocation from the cytoplasm to the plasmamembrane, which depends on the action of adaptor proteins like LAT, Gads and SLP-76 associated with the activated receptor. In a second step the regulatory residues serine 171 and threonine 165 in the activation loop of HPK1 are phosphorylated to achieve full kinase activity. A priming phosphorylation on serine 171 can be provided by protein kinase D (PKD) while threonine 165 is an autophosphorylation site.
In proliferating cells HPK1 is a specific activator of the SAPK and NFκB signaling cascades. In apoptotic cells, HPK1 is being cleaved and converted form an activator to an inhibitor of NFκB, it thereby acts as a switch priming T cells for activation-induced cell death (AICD). HPK1 may serve as an example for the capacity of distinct cellular states to evoke changes in the relative activation pattern of common lymphocyte pathways.

Fig.2: The polyomavirus-derived middleT-Antigen (PymT) assembles a large, transforming multiprotein complex at the plasmamembrane of endothelial cells. PymT is an integral membrane protein lacking intrinsic biochemical activity. Except for PymT itself the transforming complex consists of cellular signaling proteins that are recruited and activated by PymT. PymT replaces the regulatory B subunit of protein phosphatase 2A (PP2A) and binds to one of the endothelial Src family kinases (src, fyn, yes) which results in phosphorylation of the indicated tyrosines 250, 315 and 322. These phospho-tyrosine moieties can now recruit the adaptor protein ShcA, PI-3 kinase and PLCκ1. Further PH-domain containing signaling molecules like the nucleotide exchange factor SOS and the adaptor Gab are recruited via ShcA and Grb2. While SOS will activate the ras pathway, Gab contributes to persistent PI-3 kinase activation.

Fig.3: Mouse embryos that lack the cytoplasmic tyrosine kinase Syk develop lethal hemorrhages at midgestation due to aberrant shunting between the blood and lymphatic vessel system, which allows retrograde entry of blood into the developing lymphatics. The subcutaneous hemorrhages of the Syk-deficient embryo (B) outline the developing lymphatic vessels.

Lecture files

Lecture 1 - Basic principles of immunity

Lecture 2 - Innate immunity

Lecture 3 - Adaptive immunity I

Lecture 4 - Adaptive immunity II

Lecture 5 - Adaptive immunity III

Lecture 6 - Adaptive immunity IV

Lecture 7 - The Interface between Adaptive and Innate Immunity

Lecture 8 - Zytokine, Interleukine und Chemokine

Lecture 9 - Leukocytes Trafficking

Lecture 10 - Hypersensitivity

Lecture 11 - Lymphocyte Development

Lecture 11 - Autoimmunity

Lecture 12 - HIV and tumor immunology

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Reference List