Interactions between SH2 domains and phosphotyrosine sites regulate tyrosine kinase signaling networks. Selective perturbation of these interactions is challenging due to the high homology among the 120 human SH2 domains. Using an improved phage-display selection system, we generated a small antibody-mimic or ‘monobody’, termed HA4, that bound to the Abl kinase SH2 domain with low nanomolar affinity. SH2 protein microarray analysis and mass spectrometry of intracellular HA4 interactors demonstrated HA4's exquisite specificity, and a crystal structure revealed how this specificity is achieved. HA4 disrupted intramolecular interactions of Abl involving the SH2 domain and potently activated the kinase in vitro. Within cells, HA4 inhibited processive phosphorylation activity of Abl and also STAT5 activation. This work provides a design guideline for highly specific and potent inhibitors of a protein interaction domain and demonstrates their utility in mechanistic and cellular investigations.

First-generation interaction maps of Src homology 2 (SH2) domains with receptor tyrosine kinase (RTK) phosphosites have previously been generated using protein microarray (PM) technologies. Here, we developed a large-scale fluorescence polarization (FP) methodology that was able to characterize interactions between SH2 domains and ErbB receptor phosphosites with higher fidelity and sensitivity than was previously achieved with PMs. We used the FP assay to query the interaction of synthetic phosphopeptides corresponding to 89 ErbB receptor intracellular tyrosine sites against 93 human SH2 domains and 2 phosphotyrosine binding (PTB) domains. From 358,944 polarization measurements, the affinities for 1,405 unique biological interactions were determined, 83% of which are novel. In contrast to data from previous reports, our analyses suggested that ErbB2 was not more promiscuous than the other ErbB receptors. Our results showed that each receptor displays unique preferences in the affinity and location of recruited SH2 domains that may contribute to differences in downstream signaling potential. ErbB1 was enriched versus the other receptors for recruitment of domains from RAS GEFs whereas ErbB2 was enriched for recruitment of domains from tyrosine and phosphatidyl inositol phosphatases. ErbB3, the kinase inactive ErbB receptor family member, was predictably enriched for recruitment of domains from phosphatidyl inositol kinases and surprisingly, was enriched for recruitment of domains from tyrosine kinases, cytoskeletal regulatory proteins, and RHO GEFs but depleted for recruitment of domains from phosphatidyl inositol phosphatases. Many novel interactions were also observed with phosphopeptides corresponding to ErbB receptor tyrosines not previously reported to be phosphorylated by mass spectrometry, suggesting the existence of many biologically relevant RTK sites that may be phosphorylated but below the detection threshold of standard mass spectrometry procedures. This dataset represents a rich source of testable hypotheses regarding the biological mechanisms of ErbB receptors.

cis-Diaminedichloroplatinum (II) (cisplatin) is routinely used to treat various types of cancers; however, a significant number develop resistance. One of the underlying factors that contribute to cisplatin resistance is the elevated level of BCL-2 and/or BCL-XL, which promotes cell survival. A potential method of overcoming such resistance is to use a potentiator that is capable of neutralizing the antiapoptotic effects of BCL-2/BCL-XL, such as Siva-1. We previously cloned the proapoptotic protein Siva-1 and showed a possible role for it in both extrinsic and intrinsic apoptosis. Using an adenovirus-based expression system, we now show that Siva-1 can synergize with cisplatin in inducing apoptosis in MCF7 and MDA-MB-231 breast cancer cells. In an anchorageindependent clonogenicity assay, MCF7/caspase-3 cells stably expressing Siva-1, but not the control cells, showed a dramatic decrease in the number of colonies formed on one-time cisplatin treatment. Further, we show that the unique putative amphipathic helical region (SAH) in Siva-1 (amino acid residues 36-55) is necessary and sufficient for the observed enhancement in cisplatin-induced apoptosis by Siva-1. Although cisplatin treatment results in significant elevation in the expression of Fas ligand and intracellular p21 levels, expression of Siva-1 has no additional benefit. Instead, the enhancement in apoptosis seems to be due to activation of intrinsic pathway that involves caspase-9 activation. Moreover, Siva-1 augments cisplatin-mediated cell death in MCF7 cells stably expressing BCL-2. We therefore propose that Siva-1 or its SAH region can be used as a potentiator of cisplatinbased chemotherapy. (Cancer Res 2005; 65(12): 5301-9)

The human Siva gene is localized to chromosome 14q32-33 and gives rise to the full-length predominant form, Siva-1 and a minor alternate form, Siva-2 that appears to lack the proapoptotic properties of Siva-1. Our recent work has shown that the missing region in Siva-2 encodes a unique twenty amino acid putative amphipathic helical region (SAH, residues 36-55 in Siva-1). Despite the fact that Siva-1 does not belong to the BCL-2 family, it specifically interacts with the anti-apoptotic protein BCL-XL and sensitizes MCF7 breast cancer cells expressing BCL-XL to UV radiation induced apoptosis. Deletion mutagenesis has mapped the necessary region to the SAH in Siva-1. In this paper we demonstrate that the SAH region in Siva-1 is sufficient to specifically interact with the anti-apoptotic members of the BCL2 family such as BCL-XL and BCL-2 but not its apoptotic member BAX. Using transient transfections and direct microinjection of synthetic SAH peptides, we also demonstrate that the SAH region is sufficient to inhibit the BCL-XL mediated cell survival and render MDA-MB-231 and MCF7 breast cancer cells expressing BCL-XL highly susceptible to UV radiation induced apoptosis. The underlying mechanism of action of SAH mediated inhibition of BCL-XL (and/or BCL2) cell survival appears to be due to loss of mitochondrial integrity as reflected in enhanced cytochrome c release leading to the activation of caspase 9 and finally caspase 3.

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