Stephan M. Feller

c-Abl kinase regulates the protein binding activity of c-Crk.

c‐Crk is a proto‐oncogene product composed largely of Src homology (SH) 2 and 3 domains. We have identified a kinase activity, which binds to the first Crk SH3 domain and phosphorylates c‐Crk on tyrosine 221 (Y221), as c‐Abl. c‐Abl has a strong preference for c‐Crk, when compared with common tyrosine kinase substrates. The phosphorylation of c‐Crk Y221 creates a binding site for the Crk SH2 domain. Bacterially expressed c‐Crk protein lacks phosphorylation on Y221 and can bind specifically to several proteins, while mammalian c‐Crk, which is phosphorylated on tyrosine, remains uncomplexed. The protein binding activity of c‐Crk is therefore likely regulated by a mechanism similar to that of the Src family kinases. v‐Crk is truncated before c‐Crk Y221 and forms constitutive complexes with c‐Abl and other proteins. Our results suggest that c‐Abl regulates c‐Crk function and that it could be involved in v‐Crk transformation.

Structural basis for the specific interaction of lysine-containing proline-rich peptides with the N-terminal SH3 domain of c-Crk.

BACKGROUND Proline-rich segments in the guanine nucleotide exchange factor C3G bind much more strongly to the N-terminal Src homology 3 domain (SH3-N) of the proto-oncogene product c-Crk than to other SH3 domains. The presence of a lysine instead of an arginine in the peptides derived from C3G appears to be crucial for this specificity towards c-Crk. RESULTS In order to understand the chemical basis of this specificity we have determined the crystal structure of Crk SH3-N in complex with a high affinity peptide from C3G (PPPALPPKKR, Kd approximately 2 microM) at 1.5 A resolution. The peptide adopts a polyproline type II helix that binds, as dictated by electrostatic complementarity, in reversed orientation relative to the orientation seen in the earliest structures of SH3-peptide complexes. A lysine in the C3G peptide is tightly coordinated by three acidic residues in the SH3 domain. In contrast, the co-crystal structure of c-Crk SH3-N and a peptide containing an arginine at the equivalent position (determined at 1.9 A resolution) reveals non-optimal geometry for the arginine and increased disorder. CONCLUSIONS The c-Crk SH3 domain engages in an unusual lysine-specific interaction that is rarely seen in protein structures, and which appears to be a key determinant of its unique ability to bind the C3G peptides with high affinity.

SH2 AND SH3 DOMAINS AS MOLECULAR ADHESIVES : THE INTERACTIONS OF CRK AND ABL

The Src homology domains SH2 and SH3 are modular components present in many signal transduction proteins. They allow rapid formation of stable protein complexes and may also regulate protein function through intramolecular binding events. SH2 domains recognize phosphotyrosyl residues in a specific sequence context, while SH3 domains recognize a PxxP motif and additional residues that mediate binding specificity.

Ras-independent activation of the Raf/MEK/ERK pathway upon calcium-induced differentiation of keratinocytes.

MAPKs are crucially involved in the regulation of growth and differentiation of a variety of cells. To elucidate the role of MAPKs in keratinocyte differentiation, activation of ERK, JNK, and p38 in response to stimulation with extracellular calcium was analyzed. We provide evidence that calcium-induced differentiation of keratinocytes is associated with rapid and transient activation of the Raf/MEK/ERK pathway. Stimulation of keratinocytes with extracellular calcium resulted in activation of Raf isozymes and their downstream effector ERK within 10–15 min, but did not increase JNK or p38 activity. Calcium-induced ERK activation differed in kinetics from mitogenic ERK activation by epidermal growth factor and could be modulated by alterations of intracellular calcium levels. Interestingly, calcium stimulation led to down-regulation of Ras activity at the same time that ERK activation was initiated. Expression of a dominant-negative mutant of Ras also did not significantly impair calcium-induced ERK activation, indicating that calcium-mediated ERK activation does not require active Ras. Despite the transient nature of ERK activation, calcium-induced expression of the cyclin-dependent kinase inhibitor p21/Cip1 and the differentiation marker involucrin was sensitive to MEK inhibition, which suggests a role for the Raf/MEK/ERK pathway in early stages of keratinocyte differentiation.

Affinity and specificity requirements for the first Src homology 3 domain of the Crk proteins.

The specificity of SH3 domain complex formation plays an important role in determining signal transduction events. We have previously identified a highly specific interaction between the first CrkSH3 domain [CrkSH3(1)] and proline‐rich sequences in the guanine nucleotide exchange factor C3G. A 10 amino acid peptide derived from the first proline‐rich sequence (P3P4P5A6L7P8P9K10K11R12) bound with a Kd of 1.89 +/‐ 0.06 microM and fully retained the high affinity and unique selectivity for the CrkSH3(1) domain. Mutational analysis showed that P5, P8, L7 and K10 are critical for high affinity binding. A conservative mutation, K10R, significantly decreased the affinity for the CrkSH3(1) domain while increasing the affinity for Grb2. Comparative binding studies with the K10R and K10A mutant peptides to c‐Crk and v‐Crk further suggested that K10 binds via a charge‐dependent and a charge‐independent interaction to the RT loop of the CrkSH3(1) domain. Besides determining important structural features necessary for high affinity and specificity binding to the CrkSH3(1) domain, our results also demonstrate that a conservative mutation in a single amino acid can significantly alter the specificity of an SH3 binding peptide.

Interaction of Hematopoietic Progenitor Kinase 1 with Adapter Proteins Crk and CrkL Leads to Synergistic Activation of c-Jun N-Terminal Kinase

ABSTRACT Hematopoietic progenitor kinase 1 (HPK1), a mammalian Ste20-related protein kinase, is an upstream activator of c-Jun N-terminal kinase (JNK). In order to further characterize the HPK1-mediated JNK signaling cascade, we searched for HPK1-interacting proteins that could regulate HPK1. We found that HPK1 interacted with Crk and CrkL adaptor proteins in vitro and in vivo and that the proline-rich motifs within HPK1 were involved in the differential interaction of HPK1 with the Crk proteins and Grb2. Crk and CrkL not only activated HPK1 but also synergized with HPK1 in the activation of JNK. The HPK1 mutant (HPK1-PR), which encodes the proline-rich region alone, blocked JNK activation by Crk and CrkL. Dominant-negative mutants of HPK1 downstream effectors, including MEKK1, TAK1, and SEK1, also inhibited Crk-induced JNK activation. These results suggest that the Crk proteins serve as upstream regulators of HPK1. We further observed that the HPK1 mutant HPK1-KD(M46), which encodes the kinase domain with a point mutation at lysine-46, and HPK1-PR blocked interleukin-2 (IL-2) induction in Jurkat T cells, suggesting that HPK1 signaling plays a critical role in IL-2 induction. Interestingly, HPK1 phosphorylated Crk and CrkL, mainly on serine and threonine residues in vitro. Taken together, our findings demonstrate the functional interaction of HPK1 with Crk and CrkL, reveal the downstream pathways of Crk- and CrkL-induced JNK activation, and highlight a potential role of HPK1 in T-cell activation.

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.

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’.

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