Ack1: Activation and Regulation by Allostery

<div><p>The non-receptor tyrosine kinase Ack1 belongs to a unique multi-domain protein kinase family, Ack. Ack is the only family of SH3 domain containing kinases to have an SH3 domain following the kinase domain; others have their SH3 domains preceding the kinase domain. Previous reports have suggested that Ack1 does not require phosphorylation for activation and the enzyme activity of the isolated kinase domain is low relative to other kinases. It has been shown to dimerize in the cellular environment, which augments its enzyme activity. The molecular mechanism of activation, however, remains unknown. Here we present structural and biochemical data on Ack1 kinase domain, and kinase domain+SH3 domain that suggest that Ack1 in its monomeric state is autoinhibited, like EGFR and CDK. The activation of the kinase domain may require N-lobe mediated symmetric dimerization, which may be facilitated by the N-terminal SAM domain. Results presented here show that SH3 domain, unlike in Src family tyrosine kinases, does not directly control the activation state of the enzyme. Instead we speculate that the SH3 domain may play a regulatory role by facilitating binding of the MIG6 homologous region to the kinase domain. We postulate that features of Ack1 activation and regulation parallel those of receptor tyrosine kinase EGFR with some interesting differences.</p> </div

Crystallization, data collection and refinement.

<p>Crystallization, data collection and refinement.</p

Ack1 domain architecture adapted from Prieto-Echague and Miller, 2011.

<p>SAM: Sterile Alpha Motif; CRIB: Cdc42/Rac-Interactive Binding region; MHR: MIG6 Homologous Region; UBA: Ubiquitin Association region.</p

Asymmetric units of known structures of Ack1 kinase domain.

<p>(PDBIDs: 1U4D in blue, 1U46 in cyan, 1U54 in magenta, 3EQP in green and 3EQR in orange) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053994#pone.0053994-Lougheed1" target="_blank">[19]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053994#pone.0053994-Kopecky1" target="_blank">[21]</a>. (A) Non-crystallographic dimers as deposited in the PDB. (B) An alternative non-crystallographic dimer proposed to be of biological relevance.</p

Kinetic characterization of Ack1.

a<p>GST-: GST-tagged protein; P-: phosphorylated protein; CD: residues 110–476 spanning kinase domain, SH3 domain and CRIB region; KD: residues 115–389 spanning the kinase domain; KD+SH3: residues 115–453 spanning kinase and SH3 domains.</p><p>Values are averages ± SD.</p

Altered packing of the N-terminal end in the inactive state precludes dimerization and consequent activation.

<p>The region around the hydrophobic patch is highlighted for (A) active state and (B) inactive state. Same residues are highlighted in both panels. (C) Superposition of the N-terminal lobe in the active (magenta) and the inactive (green) states highlights the local reshaping of the hydrophobic patch due to the movement of the αC-helix. The area of the activated molecule involved in dimer formation is shown in surface representation. (D) Superposition of N-lobe of the inactive state (green) on the N-lobe of the molecule A (magenta) of the active dimer clearly shows that the N-terminal stretch of the protein in the inactive conformation (green) occupies the same region as that by the dimeric partner (salmon) in the activated state.</p

Allosteric activation of Ack1, EGFR and CDK.

<p>Allosteric activation via (A) symmetric dimerization of Ack1, (B) asymmetric dimerization of EGFR and (C) heterodimerization of CDK2-cyclinA. Backbone trace of each molecule of the dimeric complex is drawn, highlighting the αC helix of each kinase domain in cyan and the activation loop in blue. Molecule A of each kinase domain is shown in magenta and molecule B (cyclin in case of CDK2) in gray. For clarity, the secondary structure of only the N-terminal lobe is displayed.</p

The activated state of the Ack1 kinase domain.

<p>For clarity, secondary structure for only the N-terminal lobe is displayed. The activation loop is shown in light blue and the αC helix in cyan. Functionally important residues are highlighted.</p

Modeling of MHR interaction with Ack1.

<p>(A) Partial sequence alignment of the MIG6 sequence with its homologous region in Ack1 protein. The residue identities are highlighted. Emphasis is on the part of the MIG6 sequence that has been modeled in MIG6-EGFR complex structure <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053994#pone.0053994-Zhang2" target="_blank">[27]</a>. MIG6 residues present in the model are underlined. (B) The MIG6 fragment (corresponding to residues 805–831 of MHR) present in EGFR is modeled onto the inactive state structure of Ack1 kinase domain+SH3 domain structure. The SH3 domain is shown in surface representation. The substrate peptide binding site, as surmised from other SH3-peptide complex structures, is highlighted in deep orange shade. The MIG6 fragment, as bound to EGFR kinase domain, would present a steric conflict near its N-terminal end with the SH3 domain, as can be seen in the figure.</p

SH3 domain mediated dimer of the Ack1.

<p>(A) The kinase domains are highlighted in green and the SH3 domain in orange. Different shades are used for the individual molecules. Over 1500 Ų of surface area is buried between SH3 domains from two molecules. (B) Overlay of the Lck SH3 domain (pink) on Ack1 SH3 domain (orange). (C) Symmetric packing of two SH3 domains in two orthogonal views.</p