Role of CDK1 in Translational Regulation in the M-Phase

Cyclin dependent kinase 1 (CDK1) has been primarily identified as a key cell cycle regulator in both mitosis and meiosis. Recently, an extramitotic function of CDK1 emerged when evidence was found that CDK1 is involved in many cellular events that are essential for cell proliferation and survival. In this review we summarize the involvement of active CDK1 in the initiation and elongation steps of protein synthesis in eukaryotes. During its activation CDK1 influences the initiation of protein synthesis, promotes the activity of specific translational initiation factors and affects the functioning of a subset of elongation factors. Our review provides insights into gene expression regulation during the transcriptionally silent cell cycle/M-phase and describes quantitative and qualitative translational changes based on the extramitotic role of the cell cycle master regulator CDK1, to optimize temporal synthesis of proteins to sustain division-related processes: mitosis and cytokinesis.


Cyclin dependent kinase 1 (CDK1) is a subunit of M phase-promoting factor (MPF)
CDK1 is a key player in driving the M-phase in both meiosis and mitosis [1,2]. CDK1 activity sharply increases at the beginning of the M-phase and CDK1 is inactivated at the exit from the M-phase [3][4][5] (Figure 1).

Figure 1.
Dynamics of cyclin dependent kinase 1 (CDK1) activity, global translation and inactivation of the eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) in the M-phase. At the beginning of the M-phase the intensity of global translation is at high levels and CDK1 activity sharply increases, accompanied by nuclear envelope breakdown. CDK1 activity peaks during the assembly of the spindle and 4E-BP1 becomes hyperphosphorylated which results in its inactivation as a translational repressor. At the exit of the M-phase CDK1 activity drops and 4E-BP1 phosphorylation is lowered. The intensity of global translation decreases gradually during the time course of the M-phase. Immunoblot image using pan 4E-BP1 antibody shows an exclusive phosphorylation shift in the M-phase. Dashed green line depicts intensity of global translation; brown curved line depicts CDK1 activity; orange bars depict intensity of 4E-BP1 intensity phosphorylation in the G2, M and inter-phases. CDK1, a serine/threonine kinase, is a catalytic subunit of a complex known as MPF which is essential for cell cycle control at the G1-S and G2-M phase transitions of eukaryotic cells. CDK1 is involved in the control of such events as DNA replication and segregation, mRNA transcription, DNA repair and cell morphogenesis (reviewed in [6]). Previous studies identified several translation-associated factors as direct substrates of CDK1 in mitosis and meiosis [7,8]. The association of CDK1 with one of several cyclins is a prerequisite of CDK1 activity (Figure 1, 2). . Activated (cyclin dependent kinase 1) CDK1 supports mRNA translation during the M-phase. During the G2-phase inactive CDK1 with minimal amount of regulatory cyclin B protein is present. Prior prophase cyclin B accumulates in the nucleus and binding of cyclin B to CDK1 results in an increase of CDK1 activity. Active CDK1 promotes the nuclear lamina and nuclear envelope breakdown (NEBD). Post NEBD active CDK1 influences a number of targets including members of the translation-initiation and elongation.
In addition to cyclin binding, CDK1 activation requires dephosphorylation on Thr14 and Tyr15 residues and phosphorylation at Thr161 [9,10]. At the onset of M-phase cyclin B1 become translated and plays a fundamental role in cells entering M-phase [11]. In oocytes cyclin B1 and cyclin B2 are involved in the control of the transition from the first to the second meiotic division and cyclin B2 is able to compensate for cyclin B1 in CDK1 activation during the M-phase transition in oocyte meiosis (Li et al. 2018). A similar compensatory ability for cyclin B2/CDK1 to interact with separase has been revealed suggesting that cyclin B2/CDK1 and cyclin B1/CDK1 complexes likely function together in oocytes [13]. Cyclin B3/CDK1 complex is required for the M-phase to anaphase transition in oocytes (Li et

Global mRNA translation during the M-phase
The dynamic control mRNA translation has a great impact on many intracellular processes. According to the published data, it is considered that global translation is substantially decreased during the M-phase (Figure 1) [16][17][18][19]. Five decades ago a 50 -70% reduction of global protein synthesis in synchronized mammalian cells undergoing mitosis was described [20,21] and more recent data present a 35% decrease of translation rate during mitosis [19].
It has been documented that the intensity of protein synthesis suppression in cells undergoing mitosis is related to the method of cell synchronization [22,23]. An objection has been raised that the data of downregulation of protein translation in mitotic cells were built on the effects of cellular stress associated with cell cycle synchronization protocol (Anda and Grallert 2019). The reduction of global translation in cells undergoing cell division is believed to result from phosphorylation changes in translation initiation factors. Namely, the increased phosphorylation of an α subunit of eukaryotic initiation factor 2α (eIF2α) On the other hand oocytes isolated from mammalian ovaries are arrested in the prophase of the first meiotic division and naturally resume meiosis without chemical induction [29,30]. We and others [31,32] found that global translation decreases during meiotic progression and postfertilization. Although mitosis in synchronized cells is reported to be associated with reduced cap-dependent mRNA translation [19,21] in oocytes the capdependent translation is initiated at the resumption of meiosis as documented in in vitro matured porcine (Ellederova et al. 2006;Ellederova et al. 2008), bovine [33] and mice [17] oocytes ( Figure 1).

M-phase and reprograming of translation
Ribosomes are considered as executors of the translational program although ribosomes can also control the translation of specific mRNAs. In eukaryotes, ribosome assembly is a complex process involving more than 200 assembly factors and taking place in the nucleolus, nucleoplasm and cytoplasm (reviewed in [34]). It has been shown that CDK1 is a pronounced activator of 5'TOP mRNA translation, which includes the synthesis of all ribosomal proteins [13]. The authors considered that CDK1 possibly stimulates global translation by phosphorylating additional ribosomal proteins or proteins associated with the ribosome. CDK1 plays a role in the ribosome assembly that requires rDNA transcription, pre-rRNA processing and assembly of mature rRNAs with ribosomal proteins [13]. Ribosome assembly involves several events relying on the rDNA transcription and processing of 47S pre-rRNA into 18S, 5.8S, and 28S mature rRNAs [35,36]. Transcription of rDNA is repressed during mitosis by the CDK1-directed phosphorylation of components of the rDNA transcription machinery [37,38]. CDK1 regulates ribosome assembly by targeting specific ribosomal proteins. During the G2/M phase, CDK1 phosphorylates ribosomal protein S3 (RPS3), which is a multifunctional protein involved in translation, DNA repair, and apoptosis. Phosphorylation of RPS3 by CDK1 is important for the nuclear accumulation of RPS3 [39]. RPS3 is localized evenly in the cytoplasm of GV oocytes and with higher concentration at the newly forming spindle in NEBD oocytes and mitotic cells [40,41]. It was documented that CDK1 co-sediments in the polysome fraction, and mass spectrometry revealed that CDK1 is associated with ribosomes [42]. This finding is in line with the notion that ribosomal protein L12 (RPL12) is a known substrate of CDK1 (Table 1), and RPL12 phosphorylation was shown to enhance a mitotic translation program [43]. RPL12 has been reported to be phosphorylated on Ser38 in different species [44]. Phosphorylation of RPL12 regulates the translation of specific subsets of mRNAs during mitosis [43]. In HeLa cells phosphorylation of RPL12 at Ser38 peaks in mitosis and reaches the lowest level during the S phase [43,45]. In eukaryotes the sequence surrounding Ser38 is highly conserved and matches a consensus motif for CDK1 substrates [43]. Inhibition of CDK1 induced a progressive decrease in the percentage of heavy polysomes and a decline in polypeptide synthesis [13]. The nucleolus, the large nuclear domain assembled around ribosomal genes (rDNAs), is the site of ribosome assembly. The assembly and disassembly of the nucleolus is dependent on the equilibrium between the phosphorylation/dephosphorylation of the transcription machinery and on the pre-ribosomal ribonucleoprotein (RNP) complexes processing under the control of the CDK1 and PP1 phosphatases [46]. CDK1 regulates the activity of the nucleolar phosphoprotein nucleophosmin/B23 involved in the regulation of rRNA transcription through its histone chaperone activity [47]. B23/nucleophosmin associates with rRNA chromatin to stimulate rRNA transcription [48]. It has been shown that B23/nucleophosmin interacts with maturing ribosomal subunits and is involved in their nuclear export (Yu et al. 2006). During mitosis phosphorylation of B23/nucleophosmin by CDK1 at specific sites induces a release of B23/nucleophosmin from chromatin and inactivates its RNA binding activity [48,50]. It has been suggested that CDK1 is an obvious activator of cap 5'TOP mRNA translation including the synthesis of all ribosomal proteins [13].
Above mentioned results couple CDK1 activity and cell cycle progression to ribosome biogenesis.

CDK1 activity and localization in the cell
One insight into how one kinase can coordinate so many different events is that cyclin B1-CDK1 is targeted to different structures as the cell enters meiosis/mitosis. Cyclin B1-CDK1 is activated on centrosomes [51], and a large fraction immediately moves into the nucleus preceding the breakdown of the nuclear envelope [52,53]. Subsequently, Cyclin B1-CDK1 binds to the microtubules, to chromosomes, and to unattached kinetochores in prometaphase [8,54]. These observations indicate that the localization of Cyclin B1-CDK1 may be an important determinant of how specific substrates are recognized at specific times. In connection, we previously found that distinct translational areas are present in the oocyte post NEBD which are influenced by the mammalian target of rapamycin (mTOR)/4F axis [8,17,55].

Cap-dependent mRNA translation
Translation of mRNA is regulated mostly during the initiation phase by initiation factors interacting with a specific structure bound to the 5'UTR of an mRNA molecule, the 5' cap (m7GppN) [56]. The cap structure is specifically recognized by the eukaryotic initiation factor complex (eIF4F), comprised of the cap-binding eukaryotic translation initiation factor 4E (eIF4E), the RNA helicase eIF4A and the scaffold protein eIF4G (Sonenberg et al. 1978; Imataka, Gradi, and Sonenberg 1998). EIF4G1 also binds the poly(A)-binding protein (PABP) [59], thereby enabling the circularization of the mRNA [60].

CDK1 substitutes for mTOR control of cap-dependent translation
mTOR, a serine/threonine protein kinase, is the main regulator of 4E-BP1 activity [63], thereby releasing eIF4E and activating translation (Gingras et al. 2001). Raptor, a binding partner of mTOR, mediates the functioning of mTOR [27]. Upon inhibition of mTOR, 4E-BP1 is dephosphorylated and the affinity of 4E-BP1 for eIF4E increases (Sonenberg and Hinnebusch 2009). mTOR enhances the translation of mRNAs containing the 5' TOP motif coding for ribosomal proteins, translation-related proteins, and wider variety of proteins, such as lysosome-related proteins and metabolism-related proteins, playing pivotal roles in gene expression controls in the majority of cellular mRNAs [66,67]. It has been documented that overexpression of rapamycin-resistant mTOR mutant restored the rapamycin-inhibited activity of mTOR effectors ribosomal protein S6 kinase B1 (p70S6K) and 4E-BP1 and removed the rapamycin induced inhibition of cell cycle progression [68]. Although mTOR is considered to be the main kinase phosphorylating 4E-BP1, several other kinases are involved in the phosphorylation of various 4E-BP1 residues [69,70].
The main substrates of mTOR, p70S6K and 4E-BP1, are phosphorylated by CDK1 during mitosis [70,71] (Table 1) and CDK1 can substitute for mTOR kinase in the activation of capdependent translation in mitotic cells, suggesting that an alternate pathway for the regulation of cap-dependent translation exists [19,23,72]. It has been observed in mouse prophase lymphoblasts that CDK1 promotes mitotic growth through the increased phosphorylation of 4E-BP1 and cap-dependent protein synthesis [73].
Increased phosphorylation of 4E-BP1 occurs during meiotic M-phase in porcine, bovine and mouse oocytes (Ellederova et al. 2006;Ellederova et al. 2008;Romasko et al. 2013;Susor 2015). Although it is also diffusely located in the oocyte cytoplasm, the presence of phosphorylated 4E-BP1 at the meiotic spindle poles, kinetochores and along the polar microtubules suggests that it represents a possible means of supporting spatially localized protein production [79]. CDK1 and mTOR are the main positive regulators of 4E-BP1 phosphorylation during meiosis in mouse oocytes and CDK1 affects the activity of mTOR localized in the vicinity of chromosomes and on the MII spindle proposing that CDK1 acts indirectly on 4E-BP1 phosphorylation via mTOR activation [8].
It can be concluded that phosphorylation of 4E-BP1 promotes translation during the M-phase to support spindle assembly, highlighting the important role of CDK1 and mTOR kinase in this process.
During the M-phase CDK1 also phosphorylates and inactivates p70S6K, the other mTOR substrate [69,70]. It has been proposed that p70S6K is involved in the regulation of the TOP mRNA translation because the substrates of p70S6K include the eukaryotic translation elongation factor (eEF1A), eukaryotic elongation factor 2 (eEF2) and several ribosomal proteins [66]. Therefore, the CDK1 activity could be involved in the regulation of translation during the M-phase by decreasing the amount of translation factors available. Although p70S6K is phosphorylated by CDK1 at several sites, CDK1 does not phosphorylate p70S6K at Thr389, the mTOR phosphorylation site [70]. In HeLa cells the inhibition of CDK1 resulted in a reduced phosphorylation of the ribosomal protein S6 (RPS6), a p70S6K substrate, indicating that CDK1 supports the initiation of translation through the p70S6K signaling pathway, however, CDK1 likely does not act via mTOR as CDK1 inhibition did not alter the integrity of the cap binding complex [13].

CDK1 modulates LARP1 activity
The translation and stability of 5´TOP mRNAs can be also regulated by the mTOR dependent phosphorylation of RNA-binding La-related protein 1 (LARP1) since LARP1 functions as a repressor of ribosomal protein mRNA translation downstream of mTOR [80,81]. In LARP1-depleted mitotic Hela cells elevated levels of cyclin B and chromosomes scattered on the mitotic spindle were detected [82]. Structural analysis revealed that LARP1 recognizes the m7Gppp cap moiety and the adjacent 5´ terminal oligopyrimidine sequence found in mRNAs [83]. Binding of LARP1 to the m7Gppp cap of ribosomal protein mRNAs precludes the binding of eukaryotic initiation factor 4E (eIF4E), thus blocking the assembly of the eIF4F complex on ribosomal protein mRNAs [81,83].The activity of LARP1 is regulated in an mTOR dependent manner (Hsu et al. 2011;Yu et al. 2011). It has been reported that LARP1 phosphorylation is also dependent on the activity of CDK1 and translation of 5'TOP mRNAs is strongly enhanced by CDK1 via LARP1 [13].

CDK1 regulates the elongation step of translation
In eukaryotes, the culmination of translation initiation coincides with the formation of an 80S initiation complex in which Met-tRNAi Met is bound to the P (peptidyl) site of the ribosome. The anticodon of the Met-tRNAi Met is base-paired with the start codon of the mRNA, and the second codon of the open reading frame (ORF) is localized on the A (aminoacyl) site of the ribosome. Elongation is initiated when the cognate elongating aminoacyl-tRNA is delivered to the A site of the ribosome. The eukaryotic translation elongation factor eEF1A is activated upon binding to GTP and creates a ternary complex upon binding to aminoacyl-tRNA [86,87] .
CDK1 modulates the activity of elongation factors (Table 1). Translation elongation in eukaryotes is mediated by the determined actions of elongation factor 1A (eEF1A); elongation factor 1B (eEF1B) complex, and elongation factor 2 (eEF2). In HeLa cells and in sea urchin embryos the translation elongation rate declines in synchrony with an increase of CDK1 activity [88,89]. The eEF2 kinase (eEF2K) which inactivates eEF2 is inhibited by phosphorylation at Ser359 and CDK1 has been identified as the kinase phosphorylating eEF2K at Ser359 indicating that CDK1 stimulates the activity of eEF2 during mitosis [90] .
In human cells a conserved consensus phosphorylation site for mitotic CDK1 is present on the catalytic δ subunit of eEF1B (termed "eEF1D"). The eEF1D and eEF1Bγ subunits are physiological substrates for CDK1 during the resumption of meiosis in Xenopus oocytes [91,92].
In monkey kidney epithelial cells two eEF1D consensus CDK1 target sites were identified, Ser-133 and Thr-147, whilst eEF1D has been reported to be phosphorylated by CDK1 on Ser-133 in vitro [93]. Phosphorylation of Ser-133 during mitosis in HeLa cells is necessary for the reduced interaction of eEF1D with its substrate eEF1A and leads to a slowdown of translation elongation [87]. It has been proposed that phosphorylation of eEF1D by CDK1 leads to the reduced interaction of the catalytic subunit eEF1D with its substrate eEF1A·GDP, causing a decrease of guanine nucleotide exchange rate by the eEF1B complex followed by a lower level of active eEF1A·GTP during mitosis. Subsequently, less eEF1A·GTP is available for binding and delivering aa-tRNA to ribosomes, inducing a translational slowdown [87]. The above-mentioned data provide clear evidence of the importance of CDK1 in the regulation of elongation step of translation in eukaryotes.

Perspectives
Here we summarized findings that point to an extramitotic role of CDK1 in the cell to couple M-phase progression with protein expression. The challenge here is to experimentally uncouple the timing of M-phase progression from the extramitotic function of CDK1. Applying novel [(e.g. imaging, next generation sequencing (NGS)] methods to the naturally cycling cells will shed light to tightly coupled cellular processes. An interesting feature of CDK1-cyclin B is its localization in the cell which might be involved in the regulation of various substrates and the spatio-temporal coupling of two conserved molecular modules, CDK1-cyclin B for the cell cycle and translational regulation for gene expression. This might shed light on the physiological role on localized translation in the newly forming spindle. Future research is needed to elucidate more precisely the role of CDK1 in the regulation of mRNA translation.
Next generation sequencing of the polysomal fractions and cutting edge proteomics approach can contribute to the identification of translational changes in a positive and negative way. Additionally, detailed genome-wide analyses might reveal a subclass of transcriptome or their regulatory motives which are specifically influenced by CDK1 activity.
The direct effect of CDK1 activity on translational regulation is difficult to pinpoint because experimental manipulation of CDK1 activity might influence the timing of cell cycle progression and moreover, the number of CDK1 substrates might play an intermediate role in this process. We expect that more and more substrates with direct/indirect roles in protein synthesis will emerge in the coming years. CDK1 has an effect on NEBD which leads to the release of numerous components from the nucleoplasm which might radically effect RNA binding proteins, ribosome assembly and/or translational activity.
Further research should be oriented to the extramitotic functioning of kinases in the regulation of translation. Description of the broader role of kinases will provide a new insight into the specialized translation and translational control in human diseases such as cancer. Dysregulation of CDK1 which leads to increased cell proliferation has been identified in various cancers. Accordingly, regulation of CDK1 and usage of CDK-inhibitors have been associated with encouraging results in the treatment of cancer.