Activity of the nonreceptor tyrosine kinase Ack1 is regulated by tyrosine phosphorylation of its Mig6 homology region

Abstract

Ack1 is a proto-oncogenic tyrosine kinase with homology to the tumour suppressor Mig6, an inhibitor of the epidermal growth factor receptor (EGFR). The residues critical for binding of Mig6 to EGFR are conserved within the Mig6 homology region (MHR) of Ack1. We tested whether intramolecular interactions between the Ack1 MHR and kinase domain (KD) are regulated by phosphorylation. We identified two Src phosphorylation sites within the MHR (Y859, Y860). Addition of Src-phosphorylated MHR to the Ack1 KD enhanced enzymatic activity. Co-expression of Src in cells led to increased Ack1 activity; mutation of Y859/Y860 blocked this increase. Collectively, the data suggest that phosphorylation of the Ack1 MHR regulates its kinase activity. Phosphorylation of Y859/Y860 occurs in cancers of the brain, breast, colon, and prostate, where genomic amplification or somatic mutations of Ack1 play a role in disease progression. Our findings suggest that MHR phosphorylation could contribute to Ack1 dysregulation in tumours.

Phosphorylation of Ack1 by the Receptor Tyrosine Kinase Mer

“…The Y859F, Y860F, and double Y859F/Y860F mutants showed increased Ack1 Y284 phosphorylation compared to the wild type, as measured by Western blotting (Figure 5A). These results were consistent with the known autoinhibitory roles of these tyrosines [31]. To confirm these findings, we immunoprecipitated the Ack1 constructs and carried out Western blotting with anti-pTyr antibody (Figure 5B).…”

“…In these two peptides, the remaining tyrosine was unphosphorylated and, therefore, was available for Mer phosphorylation. Mer strongly preferred the peptide containing pY860 Ack1 contains a C-terminal regulatory region with high homology to the EGFR inhibitor Mig6 (designated the Mig6 homology region, or MHR) [31] (Figure 4A). Phosphorylation of two adjacent tyrosines (Y859 and Y860) within this region by Src increases Ack1 activity, presumably by decreasing an intramolecular autoinhibitory interaction with the catalytic domain [31].…”

“…Mer strongly preferred the peptide containing pY860 Ack1 contains a C-terminal regulatory region with high homology to the EGFR inhibitor Mig6 (designated the Mig6 homology region, or MHR) [31] (Figure 4A). Phosphorylation of two adjacent tyrosines (Y859 and Y860) within this region by Src increases Ack1 activity, presumably by decreasing an intramolecular autoinhibitory interaction with the catalytic domain [31]. We tested a series of MHR-derived peptides as in vitro substrates of Mer (Figure 4B).…”

Domain Architecture of the Nonreceptor Tyrosine Kinase Ack1

“…This Ack1-EGFR interaction may play a role in regulating Ack1 activity. Phosphorylation of the C-terminal MHR disrupts autoinhibitory interactions [ 51 , 145 ] and could lead to a conformational change that allows membrane recruitment and dimerization by the SAM domain [ 84 ]. The interplay between SH3, MHR, and Pro-rich regions is also not completely understood; it has been suggested that the interaction between the SH3 and the Pro-rich region could position the MHR for inhibiting the kinase domain [ 2 , 158 ].…”

“…The lysine that coordinates pY395 of Mig6, K879, is replaced with alanine in Ack1 (A295). Tyrosines 394–395 of Mig6 correspond to Y859-Y860 of MHR in Ack1, which are suggested to be phosphorylated similarly [ 145 ].…”