The yeast Fus1p SH3 domain recognizes non-PXXP motif targets. This is contrary to most SH3 domains, which recognize PXXP-containing sequences. In this thesis, I characterized atypical binding of the Fus1p SH3 domain and provide insight into atypical SH3 domain interactions. I demonstrated that the Fus1p SH3 domain binds R(S/T)(S/T)SL-containing peptides derived from yeast proteins, Bnr1p and Ste5p. Through mutagenesis studies and comparisons to other SH3 domains, I showed that the Fus1p SH3 domain utilizes a portion of the same binding surface as typical SH3 domains. However, the PXXP-binding surface is debilitated in the WT domain by the substitution of unusual residues at three key conserved positions. By replacing these residues, I created a version of the Fus1p SH3 domain that binds a PXXP-containing peptide with extremely high affinity. Based on my data and analysis, I clearly delineated two distinct surfaces comprising the typical SH3 domain binding interface, and show that one of these surfaces is the primary mediator of almost every “non-canonical” SH3 domain-mediated interaction. I demonstrated that the Fus1p SH3 domain also binds Bni1p and Pea2p through yeast two-hybrid experiments, which do not contain either PXXP or R(S/T)(S/T)SL motifs. Through mutagenesis studies and two-hybrid experiments, I showed that the Fus1p SH3 domain utilizes a binding surface comprised of two sub-surfaces to interact with Bni1p and Pea2p. The sub-surfaces include the same portion of the binding surface in typical SH3 domains utilized in R(S/T)(S/T)SL binding of the Fus1p SH3 domain, and a novel binding site. I also showed that the protein binding surface of the Fus1p SH3 domain has a role(s) for efficient mating. Based on my findings in this thesis, I propose that dramatic alterations in SH3 domain specificity can be simply explained as a modulation of the binding strengths of sub-binding sites within the binding surface.

Protein-protein interactions are an integral part of virtually all aspects of cellular function. Many of these interactions are mediated by small modular units called protein interaction domains (PIDs). We do not yet understand, however, how much functional information is encoded in these modules. It has previously been shown that Nbp2SH3 and Bem1SH3b domains in S. cerevisiae bind several target peptides with the same consensus sequence, yet display finely tuned affinities for each. In this study, I have shown that there exists an evolutionarily conserved ability of orthologous fungal Nbp2SH3, Bem1SH3b, and Abp1SH3 domains to discriminate between target peptides within the same species. In addition, I have developed a method to quantitatively probe SH3 domain specificity using purified SH3 domains and naturally occurring proline-rich constructs (PRRs) in the context of cell lysate from S. cerevisiae. Expansion of this work may yield valuable insights into intrinsic SH3 domain specificity.

The yeast Fus1p SH3 domain recognizes non-PXXP motif targets. This is contrary to most SH3 domains, which recognize PXXP-containing sequences. In this thesis, I characterized atypical binding of the Fus1p SH3 domain and provide insight into atypical SH3 domain interactions. I demonstrated that the Fus1p SH3 domain binds R(S/T)(S/T)SL-containing peptides derived from yeast proteins, Bnr1p and Ste5p. Through mutagenesis studies and comparisons to other SH3 domains, I showed that the Fus1p SH3 domain utilizes a portion of the same binding surface as typical SH3 domains. However, the PXXP-binding surface is debilitated in the WT domain by the substitution of unusual residues at three key conserved positions. By replacing these residues, I created a version of the Fus1p SH3 domain that binds a PXXP-containing peptide with extremely high affinity. Based on my data and analysis, I clearly delineated two distinct surfaces comprising the typical SH3 domain binding interface, and show that one of these surfaces is the primary mediator of almost every “non-canonical” SH3 domain-mediated interaction. I demonstrated that the Fus1p SH3 domain also binds Bni1p and Pea2p through yeast two-hybrid experiments, which do not contain either PXXP or R(S/T)(S/T)SL motifs. Through mutagenesis studies and two-hybrid experiments, I showed that the Fus1p SH3 domain utilizes a binding surface comprised of two sub-surfaces to interact with Bni1p and Pea2p. The sub-surfaces include the same portion of the binding surface in typical SH3 domains utilized in R(S/T)(S/T)SL binding of the Fus1p SH3 domain, and a novel binding site. I also showed that the protein binding surface of the Fus1p SH3 domain has a role(s) for efficient mating. Based on my findings in this thesis, I propose that dramatic alterations in SH3 domain specificity can be simply explained as a modulation of the binding strengths of sub-binding sites within the binding surface.

The adaptor protein p130Cas (CAS, BCAR1) represents a nodal signaling platform for integrin and growth factor receptor signaling, and influences normal development and tissue homeostasis. Its altered expression drives many pathological conditions including tumor growth, metastasis and drug resistance in many cancer types. How p130Cas contributes to many of these pathologies is still poorly understood. Therefore, the overall aim of my PhD work was to provide new insights to p130Cas signaling and its regulation. The SH3 domain is indispensable for p130Cas signaling, but the ligand binding characteristics of the p130Cas SH3 domain, and the structural determinants of its regulation were not well understood. To be able to study various aspects of p130Cas signaling we identified an atypical binding motif in p130Cas SH3 domain by establishing collaborations with Dr Veverka (Structural biology) and Dr Lepšík (Computational biochemistry; Academy of Sciences, CZ) which gave new insight into this binding interface. Through these collaborations I generated chimeras of p130Cas SH3 domain with its ligands for structural NMR analysis and learned how to visualize and analyze structures. Furthermore, my work expanded our knowledge of p130Cas SH3 ligand binding regulation and led to a novel model of Src-p130Cas- FAK...

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Protein interactions are often mediated through binding of protein interaction domains to short peptide motifs. However, how the specificity of interactions is regulated within domain families is not well understood. In this thesis, I investigated this issue through the study of two cross-reactive SH3 domains from the yeast adaptor proteins Nbp2p and Bem1p. Despite low sequence similarity and different cellular roles, the Nbp2p SH3 and Bem1p SH3b domains recognize targets containing the same consensus sequences, raising the question of how the specificity of these domains is controlled. The Bem1p SH3b domain is also unusual in containing a 40 amino acid long C-terminal extension required for folding and interaction with the Cdc42p GTPase, which I also investigated in this thesis. I demonstrated that although Nbp2p SH3 and Bem1p SH3b domains recognize the same consensus sequence, each domain possesses its own unique specificity within the context of eight yeast peptides. Through structural and mutagenesis studies, I demonstrated that the Nbp2p SH3 and Bem1p SH3b domains employ different mechanism for binding, explaining how each domain can still possess its own unique specificity, while recognizing the same consensus sequence. I found that the specificity of Bem1 SH3b domain is largely regulated through inhibitory interactions, where a conserved residue in the binding interface serves to decrease binding to non-specific targets. Most significantly I observed that reducing the specificity of Bem1 SH3b domain, without reducing its binding affinity decreases its ability to function in vivo. My structural and mutagenesis studies with the Bem1p SH3b domain also demonstrated that its C-terminal extension is involved in peptide binding surface and creates a separate surface required for interaction with Cdc42p. I also showed that the Bem1p SH3b domain is capable of simultaneously interacting with peptide targets and Cdc42p, suggesting a role for this domain in Cdc42p mediated activation. Overall, my findings demonstrated the ability of SH3 domains to perform complex functions and bind their targets with finely tuned specificity. Most importantly, I demonstrated the significance of negative interactions for specificity determination and the importance of precise specificity for in vivo function.

Protein interactions are often mediated through binding of protein interaction domains to short peptide motifs. However, how the specificity of interactions is regulated within domain families is not well understood. In this thesis, I investigated this issue through the study of two cross-reactive SH3 domains from the yeast adaptor proteins Nbp2p and Bem1p. Despite low sequence similarity and different cellular roles, the Nbp2p SH3 and Bem1p SH3b domains recognize targets containing the same consensus sequences, raising the question of how the specificity of these domains is controlled. The Bem1p SH3b domain is also unusual in containing a 40 amino acid long C-terminal extension required for folding and interaction with the Cdc42p GTPase, which I also investigated in this thesis. I demonstrated that although Nbp2p SH3 and Bem1p SH3b domains recognize the same consensus sequence, each domain possesses its own unique specificity within the context of eight yeast peptides. Through structural and mutagenesis studies, I demonstrated that the Nbp2p SH3 and Bem1p SH3b domains employ different mechanism for binding, explaining how each domain can still possess its own unique specificity, while recognizing the same consensus sequence. I found that the specificity of Bem1 SH3b domain is largely regulated through inhibitory interactions, where a conserved residue in the binding interface serves to decrease binding to non-specific targets. Most significantly I observed that reducing the specificity of Bem1 SH3b domain, without reducing its binding affinity decreases its ability to function in vivo. My structural and mutagenesis studies with the Bem1p SH3b domain also demonstrated that its C-terminal extension is involved in peptide binding surface and creates a separate surface required for interaction with Cdc42p. I also showed that the Bem1p SH3b domain is capable of simultaneously interacting with peptide targets and Cdc42p, suggesting a role for this domain in Cdc42p mediated activation. Overall, my findings demonstrated the ability of SH3 domains to perform complex functions and bind their targets with finely tuned specificity. Most importantly, I demonstrated the significance of negative interactions for specificity determination and the importance of precise specificity for in vivo function.

Protooncogene Crk was found to be upregulated in tumours with aggressive and invasive potential. The adaptor protein Crk has an important role in cell signaling: it integrates signals from activated integrins and growth factors receptors via its SH2 domain and transmits the signal to its SH3 domain binding partners that activate the small GTPases Rac1, Rap1 and Ras. This leads to regulation of cell migration, proliferation and survival. The aim of this thesis project was to inhibit the Crk dependent signaling by a competitive inhibition of the Crk SH3 domain, using a high affinity CrkSH3 binding peptoid. Binding of the inhibitor to the Crk SH3 domain prevents binding of cellular Crk SH3 interaction partners and the corresponding signal transmission is impaired. In this thesis project the effect of the Crk SH3 inhibition on the invasiveness of cancer cells was analyzed. The observed inhibitory effect on cell invasion as well as on anchorage independent growth provides a proof of therapeutical relevance of targeting CrkSH3N domain by peptoide-based inhibitors. Powered by TCPDF (www.tcpdf.org)

Protein p130Cas is the major tyrosine phosphorylated protein in cells transformed by v-crk and v-src oncogenes. P130Cas plays an important role in invasiveness and metastasis of Src-transformed cells. In breast cancer patients, high p130Cas levels are associated with higher recurrence of disease, poor response to tamoxifen treatment and lower overall survival. In non-transformed cells, after the stimulation of integrins, protein p130Cas is phosphorylated in substrate domain affecting cell migration and cytoskeletal dynamics. For this signalling is the SH3 domain of p130Cas indispensable. In this thesis, was for the first time using the Phage display method analysed and subsequently characterized the binding motif of SH3 domain of p130Cas. Based on this high-affinity motif [AP]-P-[APMS]-K-P-[LPST]-[LR]- [LPST], we predicted new interaction partners of protein p130Cas and subsequently confirmed the interaction with the Ser/Thr kinase PKN3. This kinase colocalizes with p130Cas in the nucleus and perinuclear region and could phosphorylate p130Cas. In this thesis, we also analysed the effect of phosphomimicking mutation of tyrosine from sequence ALYD, which is conserved in the sequence of SH3 domains, on ability of these domains to bind ligands. This mutation reduced binding by about 3 orders of...

P130Cas (Crk-associated substrate, CAS) is a multiadaptor protein important in integrin signalling where it positively regulates cell motility, invasion, proliferation and survival. CAS lacks enzymatic activity, but its binding to other signalling proteins could lead to the change of phosphorylation status of its substrate domain, which is the main mode, through which CAS takes part in regulating cell behavior. Local tensions in focal adhesions lead to an extension of CAS substrate domain, leaving phosphorylation sites more accessible for kinases, which subsequently leads to an increased CAS substrate domain phosphorylation. The CAS anchorage in focal adhesions is mediated by its SH3 domain, probably through the interactions with FAK, and also by C-terminal domain, where interaction partners are not known. The aim of my project is to find out, which proteins mediate the CAS anchorage to the focal adhesions. The elucidation of CAS anchorage to focal adhesions will contribute to the understanding of mechanosensory function of CAS. Experimental data suggest that tyrosine phosphorylation of the CAS SH3 domain plays an important role in the regulation of its binding properties. Another goal of my diploma project was to analyze the significance of tyrosine phosphorylation within SH3 domain and other...