Interaction of the human adenovirus proteinase with its 11-amino acid cofactor pVIc.

The interaction of the human adenovirus proteinase (AVP) and AVP-DNA complexes with the 11-amino acid cofactor pVIc was characterized. The equilibrium dissociation constant for the binding of pVIc to AVP was 4.4 microM. The binding of AVP to 12-mer single-stranded DNA decreased the K(d) for the binding of pVIc to AVP to 0.09 microM. The pVIc-AVP complex hydrolyzed the substrate with a Michaelis constant (K(m)) of 3.7 microM and a catalytic rate constant (k(cat)) of 1.1 s(-1). In the presence of DNA, the K(m) increased less than 2-fold, and the k(cat) increased 3-fold. Alanine-scanning mutagenesis was performed to determine the contribution of individual pVIc side chains in the binding and stimulation of AVP. Two amino acid residues, Gly1' and Phe11', were the major determinants in the binding of pVIc to AVP, while Val2' and Phe11' were the major determinants in stimulating enzyme activity. Binding of AVP to DNA greatly suppressed the effects of the alanine substitutions on the binding of mutant pVIcs to AVP. Binding of either or both of the cofactors, pVIc or the viral DNA, to AVP did not dramatically alter its secondary structure as determined by vacuum ultraviolet circular dichroism. pVIc, when added to Hep-2 cells infected with adenovirus serotype 5, inhibited the synthesis of infectious virus, presumably by prematurely activating the proteinase so that it cleaved virion precursor proteins before virion assembly, thereby aborting the infection.

Processive phosphorylation of p130Cas by Src depends on SH3-polyproline interactions.

Many in vivo substrates of Src family tyrosine kinases possess sequences conforming to Src homology 2 and 3 (SH2 and SH3) domain-binding motifs. One such substrate is p130Cas, a protein that is hyperphosphorylated in v-Src transformed cells. Cas contains a substrate domain consisting of 15 potential tyrosine phosphorylation sites, C- and N-terminal polyproline regions fitting the consensus sequence for SH3 domain ligands, and a YDYV motif that binds the Src SH2 domain when phosphorylated. In an effort to understand the mechanisms of processive phosphorylation, we have explored the regions of Cas necessary for interaction with Src using the yeast two-hybrid system. Mutations in the SH2 domain-binding region of Cas or the Src SH2 domain have little effect in Cas-Src complex formation or phosphorylation. However, disruption of the C-terminal polyproline region of Cas completely abolishes interaction between the two proteins and results in impaired phosphorylation of Cas. Kinetic analyses using purified proteins indicated that multisite phosphorylation of Cas by Src follows a processive rather than a distributive mechanism. Furthermore, the kinetic studies show that there are two properties of the polyproline region of Cas that are important in enhancing substrate phosphorylation. First, the C-terminal polyproline serves to activate Src kinases through the process of SH3 domain displacement. Second, this region aids in anchoring the kinase to Cas to facilitate processive phosphorylation of the substrate domain. The two processes combine to ensure phosphorylation of Cas with high efficiency.

Structural mechanism for STI-571 inhibition of abelson tyrosine kinase.

The inadvertent activation of the Abelson tyrosine kinase (Abl) causes chronic myelogenous leukemia (CML). A small-molecule inhibitor of Abl (STI-571) is effective in the treatment of CML. We report the crystal structure of the catalytic domain of Abl, complexed to a variant of STI-571. Critical to the binding of STI-571 is the adoption by the kinase of an inactive conformation, in which a centrally located "activation loop" is not phosphorylated. The conformation of this loop is distinct from that in active protein kinases, as well as in the inactive form of the closely related Src kinases. These results suggest that compounds that exploit the distinctive inactivation mechanisms of individual protein kinases can achieve both high affinity and high specificity.

Enhanced phosphorylation of Src family kinase substrates containing SH2 domain binding sites.

Src family protein-tyrosine kinases possess several modular domains important for regulation of catalytic activity and interaction with potential substrates. Here, we explore interactions between the SH2 domain of Hck, a Src family kinase, and substrates containing SH2 domain-binding sites. We have synthesized a series of peptide substrates containing a high affinity SH2 domain binding site, (phospho)Tyr-Glu-Glu-Ile. We show that the presence of this sequence in a peptide results in a dramatic increase in the phosphorylation rate of a second tyrosine located at the N terminus. Enhanced phosphorylation is not a consequence of stimulation of enzymatic activity by C-terminal tail displacement but is imparted instead by a 10-fold reduction in the Km of the phosphotyrosine-containing peptide when compared with a control. The isolated catalytic domain of the non-receptor tyrosine kinase Abl does not show a preference for the pYEEI motif-containing peptide; however, the preference is restored when the SH2 domain of Src is introduced into Abl. Furthermore, enhanced phosphorylation is dependent on the distance between SH2 domain-binding site and phosphorylatable tyrosine, with the minimum distance requirement being seven amino acids. Reversing the orientation of the pYEEI motif with respect to the substrate sequence decreases phosphorylation by down-regulated Hck, but both orientations are utilized equally well by activated Hck. We discuss the possible implications of these results for processive phosphorylation of substrates in vivo by Src family kinases.

Involvement of the alpha subunit of farnesyl-protein transferase in substrate recognition.

Using photoaffinity labeling, we have identified a region in mammalian farnesyl-protein transferase (FPTase) involved in substrate recognition. The photolabel used (Compound 1) is a peptide containing the photoactive amino acid p-benzoylphenylalanine (Bpa). Upon exposure to UV light. Compound 1 inhibits FPTase activity in a time- and concentration-dependent manner. Photoinhibition of FPTase activity by Compound 1 is prevented by adding H-Ras to the reaction mixture, indicating that labeling is targeted to the enzyme active site. We used peptide mapping by HPLC, Edman sequencing, and matrix-assisted time-of-flight (MALDI-TOF) mass spectrometry to identify the site of interaction with radiolabeled Compound 1. These experiments indicate that a specific region of the alpha subunit of the enzyme, Asp110-Arg112, is involved in substrate binding and suggest that Glu111 is likely to be the residue covalently modified by the photoaffinity label. Sequence alignments between yeast and mammalian FPTases reveal that Glu111 is conserved. The implications of this finding are discussed in light of previous mutagenesis studies on FPTase.