Christian Kardinal

Physiological signals and oncogenesis mediated through Crk family adapter proteins

The viral Crk oncogene (v‐Crk) is known to induce sarcomas in chicken and its cellular homologs c‐Crk I, c‐Crk II, and Crk‐like (CRKL) have been implicated in many signal transduction events. These include cell differentiation, cell migration, and the induced nonresponsiveness of T‐cells to stimulation of the T‐cell receptor (TCR), a state known as anergy. CRKL is also the most prominent substrate of the Bcr‐Abl oncoprotein which causes human chronic myelogenous leukemias (CML). The modular composition of the Crk family adapters which largely consist of Src homology (SH2 and SH3) domains has prompted an intensive search for physiological and pathological upstream and downstream signalling partners which selectively bind to these adapters. Upstream proteins include various receptors and large multisite docking proteins, while several protein kinases and guanine nucleotide release proteins (GNRPs) have been suggested to function downstream of c‐Crk and CRKL. Most Crk/CRKL SH2‐ and SH3‐binding proteins contain several docking sites with considerable sequence similarity. Thus the binding requirements of Crk/CRKL SH2 and SH3 domains are now well defined, providing a basis for the design of small inhibitory molecules to block the function of these adapter proteins. The enzymatic cascades activated through Crk family adapters are only partially known, but stress kinases (SAPKs/JNKs) and the GTPase Rap1, as well as the B‐Raf isoform of the Raf protein kinases, are affected in some systems. Several yet unidentified, highly selective Crk interacting proteins detectable in specific cell types remain to be studied. More detailed analyses of the enzymatic activities triggered through Crk‐type adapters will also be crucial to fully define the signalling pathways controlled by this protein family. J Cell Physiol 177:535–552, 1998.

The C-terminal SH3 domain of the adapter protein Grb2 binds with high affinity to sequences in Gab1 and SLP-76 which lack the SH3-typical P-x-x-P core motif.

The adapter Grb2 is an important mediator of normal cell proliferation and oncogenic signal transduction events. It consists of a central SH2 domain flanked by two SH3 domains. While the binding specificities of the Grb2 SH2 and N-terminal SH3 domain [Grb2 SH3(N)] have been studied in detail, binding properties of the Grb2 SH3(C) domain remained poorly defined. Gab1, a receptor tyrosine kinase substrate which associates with Grb2 and the c-Met receptor, was previously shown to bind Grb2 via a region which lacks a Grb2 SH3(N)-typical motif (P-x-x-P-x-R). Precipitation experiments with the domains of Grb2 show now that Gab1 can bind stably to the Grb2 SH3(C) domain. For further analyses, Gab1 mutants were generated by PCR to test in vivo residues thought to be crucial for Grb2 SH3(C) binding. The Grb2 SH3(C) binding region of Gab1 has significant homology to a region of the adapter protein SLP-76. Peptides corresponding to epitopes SLP-76, Gab1, SoS and other proteins with related sequences, as well as mutant peptides were synthesized and analysed by tryptophan-fluorescence spectrometry and by in vitro competition experiments. These experiments define a 13 amino acid sequence with the unusual consensus motif P-x-x-x-R-x-x-K-P as required for a stable binding to the SH3(C) domain of Grb2. Additional analyses point to a distinct binding specificity of the Grb2-homologous adapter protein Mona (Gads), indicating that the proteins of the Grb2 adapter family may have partially overlapping, yet distinct protein binding properties.

Development of highly selective SH3 binding peptides for Crk and CRKL which disrupt Crk-complexes with DOCK180, SoS and C3G

Many Src Homology 3 (SH3) domains function as molecular adhesives in intracellular signal transduction. Based on previous ultrastructural studies, short motifs which bind to the first SH3 domains of the adapters Crk and CRKL were selectively mutagenised to generate Crk/CRKL SH3-binding peptides of very high affinity and selectivity. Affinities were increased up to 20-fold compared to the best wildtype sequences, while the selectivity against a similar SH3 domain [Grb2SH3(N)] was not only retained, but sometimes increased. Blot techniques with GST-fusion peptides and in solution precipitation assays with biotinylated high affinity Crk binding peptides (HACBPs) were subsequently used to analyse the binding of these sequences to a large panel of SH3 domain-containing fusion proteins. Only those proteins which contained the CrkSH3(1) or CRKLSH3(1) domains bound efficiently to the HACBPs. A GST-HACBP fusion protein precipitated Crk and CRKL proteins out of 35S-labelled and unlabelled cell lysates. Very little binding of other cellular proteins to HACBP was detectable, indicative of a great preference for Crk and CRKL when compared to the wide variety of other endogenous cellular proteins. Moreover, HACBP disrupted in vitro preexisting Crk-complexes with DOCK180 and the exchange factors SoS and C3G, which are known targets of Crk adapters, in a concentration dependent manner. HACBP-based molecules should therefore be useful as highly selective inhibitors of intracellular signalling processes involving Crk and CRKL.

The leukaemic oncoproteins Bcr-Abl and Tel-Abl (ETV6/Abl) have altered substrate preferences and activate similar intracellular signalling pathways.

Inappropriate activation of Abl family kinases plays a crucial role in different human leukaemias. In addition to the well known oncoproteins p190Bcr-Abl and p210Bcr-Abl, Tel-Abl, a novel fusion protein resulting from a different chromosomal translocation, has recently been described. In this study, the kinase specificities of the Bcr-Abl and Tel-Abl proteins were compared to the physiological Abl family kinases c-Abl and Arg (abl related gene). Using short peptides which correspond to the target epitopes in known substrate proteins of Abl family kinases, we found a higher catalytic promiscuity of Bcr-Abl and Tel-Abl. Similar to Bcr-Abl, Tel-Abl was found in complexes with the adapter protein CRKL. In addition, c-Crk II and CRKL are tyrosine phosphorylated and complexed with numerous other tyrosine phosphorylated proteins in Tel-Abl expressing Ba/F3 cells. GTPase analysis with a RasGTP-specific precipitation assay showed constitutive elevation of GTP-loaded Ras in cells expressing the leukaemic Abl proteins. The mitogenic MAPK/Erk kinases as well as Akt/PKB, a kinase implicated to negatively regulate apoptosis, were also constitutively activated by both Bcr-Abl and Tel-Abl. The results indicate that the leukaemic Abl-fusion proteins have catalytic specificities different from the normal kinases c-Abl and Arg and that Tel-Abl is capable to activate at least some pathways which are also upregulated by Bcr-Abl.

The germinal center kinase (GCK)-related protein kinases HPK1 and KHS are candidates for highly selective signal transducers of Crk family adapter proteins.

Adapter proteins function by mediating the rapid and specific assembly of multi-protein complexes during the signal transduction which guards proliferation, differentiation and many functions of higher eukaryotic cells. To understand their functional roles in different cells it is important to identify the selectively interacting proteins in these cells. Two novel candidates for signalling partners of Crk family adapter proteins, the hematopoietic progenitor kinase 1 (HPK1) and the kinase homologous to SPS1/STE20 (KHS), were found to bind with great selectivity to the first SH3 domains of c-Crk and CRKL. While KHS bound exclusively to Crk family proteins, HPK1 also interacted with both SH3 domains of Grb2 and weakly with Nck, but not with more than 25 other SH3 domains tested. The interaction of HPK1 with c-Crk and CRKL was studied in more detail. HPK1-binding to the first SH3 domain of CRKL is direct and occurs via proline-rich motifs in the C-terminal, non-catalytic portion of HPK1. In vitro complexes were highly stable and in vivo complexes of c-Crk and CRKL with HPK1 were detectable by co-immunoprecipitation with transiently transfected cells but also with endogenous proteins. Furthermore, c-Crk II and, to a lesser extent, CRKL were substrates for HPK1. These results make it likely that HPK1 and KHS participate in the signal transduction of Crk family adapter proteins in certain cell types.

Cell-penetrating SH3 domain blocker peptides inhibit proliferation of primary blast cells from CML patients

Bcr‐Abl contributes prominently to the development of most chronic myeloid leukemias (CMLs). Prior work has identified the adapter protein CRKL as a major substrate of the Bcr‐Abl tyrosine kinase. CRKL can also bind via its first SH3 domain [SH3(1)] to specific sequences in Bcr‐Abl. Cell‐penetrating peptides were developed that bind with high affinity and selectivity to the SH3(1) domain of CRKL. They disrupt Bcr‐Abl‐CRKL complexes and strongly reduce the proliferation of primary CML blast cells and cell lines established from Bcr‐Abl‐positive patients. Activation‐specific antibodies against phosphorylated MAP kinase (MAPK) showed that MAPK activity is down‐regulated in blast cells treated with the CRKLSH3(1) blocker peptides. We conclude that the Bcr‐Abl‐CRKL complexes are largely dependent on the CRKLSH3(1) domain, that the central mitogenic cascade is down‐regulated as a consequence of the disruption of CRKLSH3(1) interactions, and that CRKL therefore contributes to the proliferation of CML blast cells.–Kardinal, C., Konkol, B., Schulz, A., Posern, G., Lin, H., Adermann, K., Eulitz, M., Estrov, Z., Talpaz, M., Arlinghaus, R. B., Feller, S. M. Cell‐penetrating SH3 domain blocker peptides inhibit proliferation of primary blast cells from CML patients. FASEB J. 14, 1529–1538 (2000)

Preparation of Disulfide-Bonded Polypeptide Heterodimers by Titration of Thio-Activated Peptides with Thiol-Containing Peptides

Abstract Titration of 2-pyridinesulfenyl (SPyr)-protected cysteine-containing peptides with thiol-unprotected peptides has proven to be a superior procedure to selectively generate disulfide-linked peptide heterodimers preventing the formation of homodimers. This procedure was used to synthesize large amounts of highly purified peptide heterodimers consisting of intracellularly active moieties, that were coupled to the third α-helix of the transcription factor Antennapedia, which serves as a ‘shuttle tag’.

An ataxia-telangiectasia-mutated (ATM) kinase mediated response to DNA damage down-regulates the mRNA-binding potential of THOC5

In response to DNA damage, transcription is blocked by inhibition of RNA polymerase II activity. The regulation of a preexisting pool of mRNAs, therefore, plays a key role in DNA repair, cell cycle arrest, or inhibition of differentiation. THOC5 is a member of the THO complex and plays a role in the export of a subset of mRNA, which plays an important role in hematopoiesis and maintaining primitive cells. Since three serine residues in the PEST domain of THOC5 have been shown to be directly phosphorylated by ataxia-telangiectasia-mutated (ATM) kinase, we examined the THOC5-dependent mRNA export under DNA damage. We show here that DNA damage drastically decreased the cytoplasmic pool of a set of THOC5-dependent mRNAs and impaired the THOC5/mRNA complex formation. The mRNP complex formed with nonphosphorylation mutant (S307/312/314A) THOC5, but not with a C-terminal deletion mutant after DNA damage, suggesting that the C-terminal domain of THOC5, but not its phosphorylation in the PEST domain, is necessary for the regulation of the mRNA-binding potency of THOC5. The cytoplasmic THOC5-dependent mRNAs were recovered by treatment with ATM kinase-specific or p53-specific siRNA, as well as by treatment with ATM kinase inhibitor, KU55933, under DNA damage conditions, suggesting that the ATM-kinase-p53 pathway is involved in this response to the DNA damage. Furthermore, the treatment with KU55933 blocked DNA damage-induced THOC5mRNP complex dissociation, indicating that activation of ATM kinase suppresses the ability of THOC5 to bind to its target mRNAs.

Tyrosine phosphatase SHP-2 is a regulator of p27Kip1 tyrosine phosphorylation

Tyrosine phosphorylation of the cell cycle regulator p27Kip1 plays a crucial role in its binding to cyclin dependent kinases and its subcellular localization. While Src and Bcr-Abl were shown to be responsible for tyrosine phosphorylation, no data are available on the dephosphorylation of p27Kip1 and the phosphatase involved. Considering the associated dephosphorylation as a pivotal event in the regulation of cell cycle proteins, we focused on the tyrosine phosphatase SHP-2, which is regulated in promyelocytic leukemia cells on G-CSF stimulation. SHP-2 was thus found in association with p27Kip1 and the G-CSF receptor, and we observed a nuclear translocation of SHP-2 on G-CSF stimulation. Using a catalytically inactive form of SHP-2 and siRNA directed against SHP-2, we could demonstrate the involvement of SHP-2 in tyrosine dephosphorylation of p27Kip1. Moreover, SHP-2 was strongly activated on G-CSF stimulation and specifically dephosphorylated p27Kip1 in vitro. Most importantly, we could illustrate that SHP-2 modulates p27Kip1 stability and contributes to p27Kip1-mediated cell cycle progression. Taken together, our results demonstrate that SHP-2 is a key regulator of p27Kip1 tyrosine phosphorylation.

Rational Development of Cell‐Penetrating High Affinity SH3 Domain Binding Peptides That Selectively Disrupt the Signal Transduction of Crk Family Adapters

CHRISTIAN KARDINAL,a GUIDO POSERN,a JIE ZHENG,b BEATRICE S. KNUDSEN,c AMGEN PEPTIDE TECHNOLOGY GROUP,d ISMAIL MOAREFI,e AND STEPHAN M. FELLERa,f aLaboratory of Molecular Oncolology, MSZ, University of Würzburg, Würzburg, Germany bDepartment of Structural Biology, St. Jude Childrens Hospital, Memphis, Tennessee cDepartment of Pathology, New York Hospital, New York, New York dBoulder, Colorado eMax Planck Institute for Biochemistry, Martinsried, Germany