Constitutively active NDR1-PIF kinase functions independent of MST1 and hMOB1 signalling
Introduction
Most signal transduction cascades transmit signals through protein kinases, which consequently represent one of the largest superfamilies found in the human genome [1]. In particular members of the AGC (protein kinase A (PKA)/PKG/PKC-like) subfamily of protein kinases have crucial cellular functions in cell growth, metabolism, proliferation and survival [2]. All AGC kinases share structural similarities, and many AGC kinases require phosphorylation of two conserved regulatory sites for activation: one conserved Ser/Thr residue within the activation segment (also termed T-loop) and one within the C-terminal hydrophobic motif (HM). As exemplified by the biochemical and structural characterization of the AGC kinase, Akt/PKB [3], [4], both regulatory sites must be phosphorylated simultaneously to achieve full kinase activation.
The NDR (nuclear Dbf2-related)/LATS (large tumour suppressor) kinase family is a subgroup of AGC kinases and consists of four related serine/threonine protein kinases (NDR1/STK38, NDR2/STK38L, LATS1, and LATS2) in the human genome [1], [5]. In mammals, LATS1/2 kinases are central to the Hippo tumour suppressor pathway [6], [7], [8], [9], and NDR kinases regulate essential processes, such as centrosome duplication [10], [11], cell cycle/mitotic progression [12], [13], [14], [15], ciliogenesis [16], neuronal dendrite/synapse formation [17], and apoptotic signalling [18], [19], where the latter function appears to be important for suppression of tumour growth [20]. In spite of the rapid progress in deciphering functions of mammalian LATS1/2, the mechanism of NDR regulation by phosphorylation must serve as a model for LATS regulation [8]. Therefore, studies addressing the regulatory mechanisms of NDR kinases are also very relevant for understanding the regulation of mammalian LATS-Hippo signalling.
Previously, it was reported that human NDR1 is dramatically activated upon inhibition of protein phosphatase 2A by okadaic acid (OA) [21], [22], which is not surprising since NDR1, like many other AGC kinases, must be phosphorylated on Ser281 (T-loop phosphorylation) and Thr444 (HM phosphorylation) for full activation [21], [22], [23], [24], [25]. However, NDR kinases are unique among AGC kinases because they have a distinctive activation mechanism. Competitive binding of the co-activator hMOB1 or the inhibitor hMOB2 to a conserved N-terminal regulatory (NTR) domain of NDR1 regulates kinase activity [23], [24], [26]. Initially, it was thought that binding of hMOB1 to the NTR only stimulates Ser281 autophosphorylation, but recent evidence suggests that hMOB1/NDR complex formation plays also a role in HM phosphorylation of NDR1, since Thr444 phosphorylation by MST1 is hMOB1 dependent in human cells [10], [27]. Therefore, NDR1 is regulated by a multistep process involving HM phosphorylation on Thr444, autophosphorylation of Ser281, and NTR binding of hMOB1. However, the order and dependency of these regulatory events are currently unknown [8].
Here, we dissected these three signalling steps by examining the basal and OA-stimulated activities of NDR1 variants carrying modifications that mimic HM phosphorylation and/or abolish hMOB1/NDR1 complex formation. These efforts revealed that NDR1-PIF, an NDR1 variant containing the PRK2 hydrophobic motif, is hyperactive and maintains high levels of Ser281 autophosphorylation despite loss of hMOB1 binding. Moreover, by studying NDR1-PIF in context of the previously reported MST1/hMOB1/NDR1 centrosome duplication signalling cascade [10], we demonstrate that expression of centrosome targeted and hMOB1-binding deficient NDR1-PIF is sufficient to restore centrosome amplification in MST1-depleted cells, thereby showing that NDR1-PIF can function independent of hMOB1 and MST1 signalling in human cells.
Section snippets
Cell culture and transfections
Cos-7, U2-OS and PT67 cells were maintained in DMEM supplemented with 10% foetal calf serum. Exponentially growing Cos-7 cells were plated at a consistent confluence (1 × 106 cells/10-cm dish) and transfected the next day using Fugene 6 (Roche) or Metafectene Pro (Biontex) as described by the manufacturer. U2-OS cells were transfected with Lipofectamine 2000 (Invitrogen) as described by the manufacturer.
Cell treatments
In OA experiments, cells were treated for 60 min with 1 μM okadaic acid (OA; Enzo Life Sciences).
PIFtide based hydrophobic motif (HM) modifications in human NDR1 result in hyperactive NDR1 kinases, while phospho-mimetic T444D and T444E mutations do not
To develop a research tool allowing the examination of the importance of HM phosphorylation of human NDR1, we characterised NDR1 variants carrying different HM modifications (Fig. 1, Fig. 2). Like the majority of AGC kinases, NDR1 is stimulated by C-terminal HM phosphorylation on Thr444 phosphorylation [2], [5]. To test whether Asp/Glu substitutions at Thr444 can mimic Thr444 phosphorylation, thereby possibly generating constitutively active NDR1, we generated NDR1 mutants carrying Thr444 to
Discussion
Taken together, our study provides novel insights into the regulatory mechanisms of human NDR1 kinase and the role of NDR1 in centrosome duplication. PIFtide based HM modifications of NDR1 result in increased kinase activities (Fig. 1, Fig. 2), which cannot be explained by altered binding of NDR1 to the co-activator hMOB1 or the inhibitor hMOB2 (Fig. 4, Fig. 5). The analysis of hMOB1 binding deficient NDR1-PIF further demonstrated that NDR1-PIF remains active independent of hMOB1/NDR1-PIF
Conclusion
In summary, our data presented in this study provide, for the first time, evidence suggesting that, once NDR1 has been modified to mimic Thr444 phosphorylation on the HM, Ser281 autophosphorylation of NDR1 can occur independent of complex formation with hMOB1. NDR1-PIF efficiently mimics Thr444 phosphorylation, remaining active even in the absence of hMOB1/NDR1-PIF complex formation. In human cells, centrosome-enriched and hMOB1-binding deficient NDR1-PIF compensates for NDR1- or MST1-depletion
Conflict of interest
The authors declare that they have no conflict of interest.
Acknowledgements
We thank J. Lisztwan and R. Jeroen Pasterkamp for careful reading of the manuscript, and all members of our laboratory for helpful discussions. This work was supported by the AICR grant 11-0634, the BBSRC grant BB/I021248/1 and the Wellcome Trust grant 090090/Z/09/Z, and supported by the National Institute for Health Research University College London Hospitals Biomedical Research Centre (grant HGJA). A.H. is a Wellcome Trust Research Career Development fellow at the UCL Cancer Institute.
References (36)
- et al.
Mol. Cell
(2002) - et al.
Semin. Cell Dev. Biol.
(2012) - et al.
Curr. Biol.
(2009) - et al.
Mol. Cell
(2007) - et al.
Curr. Biol.
(2009) - et al.
Curr. Biol.
(2010) - et al.
Neuron
(2012) - et al.
Curr. Biol.
(2008) - et al.
J. Biol. Chem.
(1999) - et al.
J. Biol. Chem.
(2003)
J. Biol. Chem.
Cell. Signal.
J. Biol. Chem.
Curr. Biol.
Curr. Opin. Neurobiol.
Neuron
Science
Nat. Rev. Mol. Cell Biol.
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