Constitutively active NDR1-PIF kinase functions independent of MST1 and hMOB1 signalling

• Published in: Cellular signalling Vol. 26; no. 8; pp. 1657 - 1667
• Main Authors: Cook, Dorthe, Hoa, Lily Y., Gomez, Valenti, Gomez, Marta, Hergovich, Alexander
• Format: Journal Article
• Language: English
• Published: England Elsevier Inc 01-08-2014
• Subjects: Adaptor Proteins, Signal Transducing - metabolism; Amino Acid Sequence; Amino Acid Substitution; Animals; Binding; Biochemistry; Cell Cycle Proteins - antagonists & inhibitors; Cell Cycle Proteins - genetics; Cell Cycle Proteins - metabolism; Cell Line; Cellular; Centrosome - metabolism; Centrosome duplication; Chlorocebus aethiops; Complex formation; COS Cells; Humans; Hydrophobic motif phosphorylation; Intracellular kinase signalling; Intracellular Signaling Peptides and Proteins - antagonists & inhibitors; Intracellular Signaling Peptides and Proteins - genetics; Intracellular Signaling Peptides and Proteins - metabolism; Kinases; MOB proteins; Molecular Sequence Data; MST1/STK4; NDR1/STK38; Phosphorylation; Protein Binding; Protein Serine-Threonine Kinases - metabolism; Proteoglycans - metabolism; Reproduction; RNA Interference; RNA, Small Interfering - metabolism; Signal Transduction; Signalling; Intracellular kinase signalling; MOB; Hydrophobic motif phosphorylation; HM; MST1; STK; MST1/STK4; PRK2; PIF; NDR1; Centrosome duplication; MOB proteins; LATS; NDR1/STK38
• ISSN: 0898-6568; 1873-3913
• DOI: 10.1016/j.cellsig.2014.04.011

The human MST1/hMOB1/NDR1 tumour suppressor cascade regulates important cellular processes, such as centrosome duplication. hMOB1/NDR1 complex formation appears to be essential for NDR1 activation by autophosphorylation on Ser281 and hydrophobic motif (HM) phosphorylation at Thr444 by MST1. To dissect these mechanistic relationships in MST1/hMOB1/NDR signalling, we designed NDR1 variants carrying modifications that mimic HM phosphorylation and/or abolish hMOB1/NDR1 interactions. Significantly, the analyses of these variants revealed that NDR1-PIF, an NDR1 variant containing the PRK2 hydrophobic motif, remains hyperactive independent of hMOB1/NDR1-PIF complex formation. In contrast, as reported for the T444A phospho-acceptor mutant, NDR1 versions carrying single phospho-mimicking mutations at the HM phosphorylation site, namely T444D or T444E, do not display increased kinase activities. Collectively, these observations suggest that in cells Thr444 phosphorylation by MST1 depends on the hMOB1/NDR1 association, while Ser281 autophosphorylation of NDR1 can occur independently. By testing centrosome-targeted NDR1 variants in NDR1- or MST1-depleted cells, we further observed that centrosome-enriched NDR1-PIF requires neither hMOB1 binding nor MST1 signalling to function in centrosome overduplication. Taken together, our biochemical and cell biological characterisation of NDR1 versions provides novel unexpected insights into the regulatory mechanisms of NDR1 and NDR1's role in centrosome duplication.
•NDR1-PIF, an NDR1 variant containing the PRK2 hydrophobic motif, is hyperactive.•NDR1-PIF remains active independent of hMOB1/NDR1-PIF complex formation.•Ser281 autophosphorylation of NDR1-PIF occurs independent of hMOB1 binding.•NDR1-PIF requires neither hMOB1 nor MST1 to function in centrosome duplication.•Centrosome-targeted NDR1-PIF is sufficient to compensate for NDR1 or MST1 depletion.