Multisite phosphorylation is certainly a common pathway to modify protein function
Multisite phosphorylation is certainly a common pathway to modify protein function interaction and activity design kinase response in physiological conditions. these strategies using the above-mentioned complementary MS strategies Fadrozole allows the mechanistic analysis of systems with an increase of complicated phosphorylation and relationship patterns. Therefore we set out to characterize the phosphorylation-induced activation of Polo-like kinase 1 (Plk1) by full-length Aurora kinase A (Aur-A) and its protein cofactor Bora a complex and biologically relevant three protein system in which the role of multisite phosphorylation remains to be deciphered. Plk1 activation is essential for cells to enter mitosis after recovery from a DNA damage-induced cell cycle arrest as shown by a wide range of studies.30?33 While the cytophysiological implications of Plk1 activation have been described in detail its molecular mechanism is still poorly understood. To date it is only known that Plk1 is usually activated through the Aur-A-catalyzed phosphorylation of Thr210 and that this process is usually augmented by the Aur-A activator Bora (Physique ?Physique11B).32 33 Bora is a largely disordered protein that becomes multiply phosphorylated by several kinases.34?36 Its N-terminal domain name is a stable conversation partner Fadrozole and a phosphorylation substrate of Aur-A 37 suggesting that Aur-A activation involves the Bora N-terminus. Interestingly the Bora N-terminus can also be bound and phosphorylated by Plk1.31 Neither the specific sites nor the functions of these N-terminal Bora phosphorylations have yet been characterized. Additionally Plk1 can phosphorylate Bora at its C-terminal residues Ser497 and Ser501 resulting in Bora Fadrozole degradation.31 38 This degradation course of action will not be studied since it commences after the Fadrozole initial Aur-A/Bora-mediated Plk1 activation. Here we aim to characterize the complex interplay of Plk1 Aur-A and the Bora N-terminus (residues 1-150 from here on termed BoraNT) in mechanistic detail specifically focusing on the implications of the BoraNT phosphorylation status its phosphorylatable sites and the sequential interactions among the three proteins. Using native MS and cross-linking-MS we demonstrate that Aur-A/BoraNT complex formation is usually independent of the BoraNT phosphorylation state. In contrast substantial Plk1/BoraNT complex formation depends on the considerable Plk1- and Aur-A-catalyzed multisite phosphorylation of BoraNT with more than 75% of its Ser and Thr residues being phosphorylated. With top-down and quantitative bottom-up proteomics methods this BoraNT multisite phosphorylation could be fully characterized at the amino acid residue level. Pushing the limits of top-down proteomics we localize up to 16 BoraNT phosphorylation sites and define the order by which Plk1 and Aur-A process these substrate sites. By means of IMS-MS Rabbit Polyclonal to DNA Polymerase alpha. we observe that these phosphorylation events induce a significant conformational switch of BoraNT providing a rationale for the correlation between BoraNT multisite phosphorylation and enhanced Plk1/BoraNT complex formation. Together the complementary mass spectrometric data give detailed molecular insights into the Aur-A/Bora/Plk1 reaction mechanism emphasizing the potential of our integrated MS approach to comprehensively describe the mechanistic principles and structural effects of Fadrozole multisite phosphorylation. Results Time-Resolved Native MS Analysis Elucidates the Interplay between Multisite Phosphorylation and Stable Interactions in the Tripartite Aur-A/BoraNT/Plk1 System Our MS-based strategy is centered on high-resolution Fadrozole native Orbitrap MS as it has an accurate readout for any phosphorylation occasions at the proteins and proteins complicated level. Previously it has allowed us to qualitatively verify stable complicated formation between your Aur-A kinase domains and BoraNT while concurrently following Aur-A-catalyzed BoraNT phosphorylation.18 To probe the interplay of Aur-A BoraNT and Plk1 we employ high-resolution native MS so which allows us to monitor not merely protein phosphorylation states but also the relative abundances of rising noncovalent protein complexes (find Supporting Information). Consultant indigenous mass spectra attained for an assortment of Aur-A BoraNT and Plk1 before and after incubation with Mg-ATP (find Amount S1 for sequences and domains architecture from the three proteins) are proven in Amount.