Human NDR kinases control G1 progression/S-phase entry by regulating p21 and c-myc stability

Hauke Cornils1*, Reto S. Kohler1, Alexander Hergovich1 and Brian A. Hemmings1*

1 _{Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, CH-4058}

Basel, Switzerland

*_{Corresponding authors at the above address:}

Brian A. Hemmings

Tel: +41-61-6974872; Fax: +41-61-6973976; Email: [email protected] Hauke Cornils

3.2.1 Abstract

The G1-phase of the cell cycle is an important integrator of internal and external cues, allowing a cell to decide whether to proliferate, differentiate or die. We identify here a role for mammalian NDR kinases in regulating G1-progression and S-phase entry. Although characterized well terms of biochemical regulation and upstream signaling, signaling downstream of mammalian NDR kinases remains largely unknown. Here we describe downstream signaling mechanisms by which NDR kinases regulate cell cycle progression. NDR kinases directly impact on the protein stability of the c-myc proto-oncogene and the Cyclin-Cdk inhibitor protein p21. Addressing the mechanisms behind the regulation of c-myc and p21 stability, we identify p21 as the first in vivo substrate for mammalian NDR kinases. Whereas p21 levels are regulated in a kinase activity dependent manner, c-myc levels are regulated independently of NDR kinase activity: NDR kinases bind to c-myc and interfere with c-myc ubiquitination and degradation.

3.2.2 Introduction

In order to duplicate, cells have to proceed through a defined order of events collectively called the cell cycle. The eukaryotic cell cycle consists of 4 phases, which can roughly be defined by growth and preparation for the duplication of the genetic material in G1-phase, duplication of the genetic material in S-phase, preparation for separation in G2-phase and finally separation of the genetic material into two daughter cells in M-phase. Proper progression through a given cell cycle phase and unidirectional transition between the phases are highly controlled on multiple levels (1). Defects in the mechanisms controlling the cell cycle have been shown to result in accumulation of genetic alterations and subsequent cancer development (2). The G1- phase of the cell cycle is a very important integrator of internal and external cues, allowing cells to grow, process outside information or repair damage before entering S-phase. In G1 a cell decides whether to self renew, differentiate or die (1). Entry into S-phase is mediated by the action of Cyclin-Cdk complexes. Initially Cyclin D- Cdk4/6 and later Cyclin E-Cdk2 complexes phosphorylate the Rb tumor suppressor protein allowing dissociation of Rb from E2F transcription factors and subsequent transcription of genes required for S-phase entry (3).

The activity of Cyclin-dependent kinases is controlled on multiple levels (4). The association of Cdks with Cylin subunits is a prerequisite for Cdk activation. This process is controlled firstly by the availability of the Cyclin sub-unit, which abundance is regulated both by transcriptional and post-transcriptional processes (4). Furthermore, Cyclin-Cdk inhibitor (CKI) proteins of the Cip/Kip and INK4 family control Cyclin-Cdk activity by different mechanisms. Whereas members of the Cip/Kip family such as p21, p27 and p57 directly inhibit the Cdk activity, the INK4 family members p15, p16, p18 and p19 actively promote Cyclin-Cdk complex

disassembly. Interestingly, the Cip/Kip family members p21 and p27 are needed for efficient assembly of Cyclin D-Cdk4/6 complexes (5). Importantly, loss of several CKI proteins has been associated with tumor development (2).

Members of the NDR family of Ser/Thr kinases are highly conserved from yeast to men and have been implicated in the regulation of a variety of biological processes (6). With the regulation of mitotic exit, cell growth, proliferation, centrososme duplication and morphogenesis, NDR kinases across species have been shown to function in processes tightly linked to the cell cycle (6). The human genome encodes four different NDR kinase family members: NDR1/2 and LATS1/2 (7). The kinases LATS1/2 function as part of the HIPPO pathway thereby controlling the localization and function of the YAP oncogene (8). The HIPPO pathway thereby controls cell growth, cell size, proliferation and apoptosis. Furthermore, roles for LATS1 and LATS2 in controlling mitotic exit and genomic stability have been described (9, 10). Although well characterized in terms of biochemical regulation, functions for the other two NDR family kinases in the human genome NDR1 and NDR2 only recently started to unravel. In cellular systems NDR kinases have been implicated in the regulation of centrosome duplication, apoptosis and the alignment of mitotic chromosomes (11-13). Furthermore, a recent study indicated a tumor suppressive function for NDR1/2 in mice by controlling proper apoptotic responses (Cornils, et al. submitted). Interestingly, with the involvement of MST1 and hMOB1 in the regulation of NDR1/2 in centrosome duplication and apoptosis and MST2 in the alignment of mitotic chromosomes, several components of the HIPPO pathway also function in the regulation of NDR1/2 (11, 13, 14). However, although first functions for NDR1/2 were defined recently, downstream signaling remained elusive. Furthermore, although NDR kinases have been implicated in cell-cycle dependent

processes such as centrosome duplication and the alignment of mitotic chromosomes, discrepancies exist whether NDR kinases are activated in M or S-phase of the cell cycle (11, 14). Here we addressed the activation of NDR1/2 throughout the cell cycle. We show that NDR1/2 are activated in G1-phase by MST3, the third MST-family kinase shown to function upstream of NDR1/2. Even more importantly, with the direct regulation of c-myc and p21 stability, we define first downstream signaling mechanisms by which NDR kinases control G1-progression and S-phase entry.

3.2.3 Results

NDR kinases are activated in G1-phase by MST3

Earlier reports have implicated NDR family kinases in the regulation of cell-cycle dependent processes such as mitotic chromosome alignment and the regulation of centrosome duplication (11, 14). However it has been reported that the activation of NDR kinases can take place in M-phase or in S phase of the cell-cycle. In order to address this discrepancy we analyzed NDR activity changes during the cell-cycle. M- phase arrested HeLa S3 cells were washed free from nocodazole, replated in fresh medium and followed for up to 18h after release (Figure 1A). NDR kinase activation was assessed using an antibody specific for the hydrophobic motif (HM) phosphorylation of NDR1 and NDR2 (referred to as anti-T444P). HM- phosphorylation of NDR1/2 was nearly absent in M-phase, but increased after 3h and peaked around 6-8h after release. The activation of NDR1/2 coincided with accumulation of the G1-phase marker CyclinD1 and PI-staining confirmed that 4-6h after release the cells were mainly in G1. HM-phosphorylation of NDR1/2 started to gradually decrease after 12h up to the end of the experiment, which coincided with the accumulation of the S/G2-phase marker CyclinA. Thus, NDR kinases were activated

Figure 1. NDR kinases are activated by MST3 in G1-phase of the cell-cycle.