Marita Hiipakka

Identification of preferred protein interactions by phage‐display of the human Src homology‐3 proteome

We have determined the human genome to contain 296 different Src homology‐3 (SH3) domains and cloned them into a phage‐display vector. This provided a powerful and unbiased system for simultaneous assaying of the complete human SH3 proteome for the strongest binding to target proteins of interest, without the limitations posed by short linear peptide ligands or confounding variables of more indirect methods for protein interaction screening. Studies involving three ligand proteins, human immunodeficiency virus‐1 Nef, p21‐activated kinase (PAK)2 and ADAM15, showed previously reported as well as novel SH3 partners with nanomolar affinities specific for them. This argues that SH3 domains may have a more dominant role in directing cellular protein interactions than has been assumed. Besides showing potentially important new SH3‐directed interactions, these studies also led to the discovery of novel signalling proteins, such as the PAK2‐binding adaptor protein POSH2 and the ADAM15‐binding sorting nexin family member SNX30.

Activation of NFAT-Dependent Gene Expression by Nef: Conservation among Divergent Nef Alleles, Dependence on SH3 Binding and Membrane Association, and Cooperation with Protein Kinase C-θ

ABSTRACT Here we show that the potential to regulate NFAT is a conserved property of different Nef alleles and that Nef residues involved in membrane targeting and SH3 binding are critical for this function. Cotransfection of an activated protein kinase C-θ (PKC-θ) with Nef implicated PKC-θ as a possible physiological cofactor of Nef in promoting NFAT-dependent gene expression and T-cell activation.

Versatile retargeting of SH3 domain binding by modification of non-conserved loop residues

Src‐homology (SH3) domain belongs to a class of ubiquitous modular protein domains found in nature. SH3 domains have a conserved surface that recognises proline‐rich peptides in ligand proteins, but additional contacts also contribute to binding. Using the SH3 domain of hematopoietic cell kinase as a test case, we show that SH3 binding properties can be profoundly altered by modifications within a hexapeptide sequence in the RT‐loop region that is not involved in recognition of currently known consensus SH3 target peptides. These results highlight the role of non‐conserved regions in SH3 target selection, and introduce a strategy that may be generally feasible for generating artificial SH3 domains with desired ligand binding properties.

Capacity of simian immunodeficiency virus strain mac Nef for high-affinity Src homology 3 (SH3) binding revealed by ligand-tailored SH3 domains.

The simian immunodeficiency virus (SIV) Nef protein contains a consensus Src-homology 3 (SH3) binding motif. However, no SH3-domain proteins showing strong binding to SIV Nef have yet been found, and its potential capacity for high-affinity SH3 binding has therefore remained unproven. Here we have used phage-display-assisted protein engineering to develop artificial SH3 domains that bind tightly to SIV strain mac (SIVmac) Nef. Substitution of six amino acids in the RT loop region of Hck-SH3 with the sequence E/DGWWG resulted in SH3 domains that bound in vitro to SIVmac Nef much better than the natural Hck- or Fyn-SH3 domains. These novel SH3 domains also efficiently associated with SIVmac Nef when co-expressed in 293T cells and displayed a strikingly differential specificity when compared with SH3 domains similarly targeted for binding to human immunodeficiency virus type 1 (HIV-1) Nef. Thus, SIVmac Nef is competent for high-affinity SH3 binding, but its natural SH3 protein partners are likely to be different from those of HIV-1 Nef.