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. 2023 Oct 31;34(12):pe5. doi: 10.1091/mbc.E22-06-0196

CDK signaling via nonconventional CDK phosphorylation sites

Ervin Valk a, Mihkel Örd a, Ilona Faustova a, Mart Loog a,*
Editor: Doug Kelloggb
PMCID: PMC10846619  PMID: 37906435

Abstract

Since the discovery of cyclin-dependent kinases (CDKs), it has been perceived as a dogma that CDK signaling in the cell cycle is mediated via targeting the CDK consensus sites: the optimal and the minimal motifs S/T-P-x-K/R and S/T-P, respectively. However, more recent evidence suggests that often the CDK phosphorylation events of regulatory importance are mediated via nonconventional CDK sites that lack the required +1Pro of the consensus site motif. In these cases, the loss of specificity seems to be compensated via distant docking interactions facilitated by 1) phosphorylated priming sites binding to phospho-adaptor Cks1 and/or 2) cyclin-specific docking interactions via Short Linear Motifs (SLiMs) in substrates. This Perspective discusses the possible reasons why nonconventional CDK sites are used for CDK signaling. First, the nonconventional CDK sites can act as specificity filters to recognize and distinguish the CDK signal from many other proline-directed kinases in cells. Second, the nonconventional CDK sites in combination with the docking mechanisms provide a much wider range of phosphorylation rates, and thus, also a wider range of CDK thresholds during the accumulation and decline of CDK activity during the cell cycle. As a large number of Cks1-dependent nonconventional CDK sites have been discovered recently, past studies focusing on mutating only the consensus sites should likely be critically reexamined. It is also very likely that phosphorylation of nonconventional sites is crucial in many other kinase-signaling networks.

INCREASING EVIDENCE OF nonconventional SITES IN CDK SIGNALING

The cyclin-dependent protein kinases (CDKs) are master regulators of the eukaryotic cell division cycle (Morgan, 2007). These enzymes phosphorylate thousands of serine and threonine residues in hundreds of target proteins to trigger and temporally order the complex processes of cell duplication. So far, the majority of studies over the past decades have been guided by the known CDK consensus phosphorylation motifs: T/S-P+1-X-K/R+3, with a proline in position +1 and a basic residue in position +3 from the phosphorylated residue. The minimal consensus site is considered to be just the S/T-P (Brown et al., 1999, 2015; Mok et al., 2010). However, by the 1990s, several proteins, including vimentin, desmin, and myosin II, were found to be phosphorylated by Cdk1 on S/T residues that were not followed by a P+1 (Chou et al., 1991; Satterwhite et al., 1992; Kusubata et al., 1993). Recently, it has become more evident that a considerable fraction of key CDK phosphorylation sites may not bear the consensus sequence at all. Recently, several proteome-wide studies have hinted at a much larger role of nonconventional CDK sites in CDK signaling than previously anticipated (Michowski et al., 2020; Al-Rawi et al., 2023).

SUBSTRATE SPECIFICITY IN PROTEIN PHOSPHORYLATION

The substrate specificity of protein kinases is controlled at multiple levels of the enzyme–substrate interaction: 1) substrate consensus motif binding to the kinase active-site region; 2) specific interactions between docking motifs in substrates and docking pockets in kinase complexes; and 3) proteins that are not functional parts of the kinase complexes, but can bring the kinase and its substrate to proximity in the cellular environment, or on the surface of large protein complexes and organelles. The conventional consensus site is a Short-Linear Motif (SLiM) whose amino acid side chain chemistries at key positions mediate the binding configuration that facilitates the phosphoryl transfer of the gamma phosphate of ATP to the hydroxyl group of serine, threonine, or tyrosine side chains in a target protein. The key constant describing the enzyme specificity towards its substrates is kcat/KM. The kcat/KM value can be defined as the rate constant of an enzyme reaction under conditions of limiting substrate concentration ( [S] << KM, where velocity v0 = kcat/KM [E] [S] ). Relative individual rates of catalysis in a mix of different substrates are directly proportional to their kcat/KM[S] values at any range of substrate concentrations. Different specificities of kinase substrates can be compared as relative values of kcat/KM, as the phosphorylation specificity does not depend only on substrate affinity, but also on the rate of the catalytic phosphoryl-transfer step (Lim et al., 2015). This constant, also called “the specificity constant” is a measure of how efficiently an enzyme converts substrates into products. Thus, even when substrates have similar affinities to the enzyme, they may have different specificities, if their catalytic steps have different rates. The same can be also true for the opposite: different substrate affinities can yield similar specificities.

HOW ARE THE CONSENSUS SITE REQUIREMENTS BYPASSED IN THE CASE OF NONCONVENTIONAL CDK SITES?

Very often, the experimentally detected phosphorylation sites do not contain a consensus motif or have only a partial version of it. In these cases, to compensate for the absence of the consensus motif additional specificity elements may come into play. For example, in the case of a 62-amino acid long substrate peptide phosphorylation assays with Cyclin B/CDK1 complex, a nonconventional phosphorylation site without the P+1 was quite efficiently phosphorylated when a cyclin docking motif RXL, located at a distance of 17 amino acids downstream, was present (Brown et al., 2015). Similarly, it was shown that the S-phase cyclin-specific docking motif NLxxxL helps to phosphorylate a nonconventional CDK site S91 in Far1 of Saccharomyces cerevisiae (Faustova et al., 2021). This phosphorylation event creates a di-phosphodegron, which is a short protein sequence motif whose phosphorylated form binds a ubiquitin ligase to label the protein with a degradation signal – ubiquitin chains. Another well-studied example is the CDK inhibitory kinase Wee1, whose S. cerevisiae orthologue Swe1 gets phosphorylated at many nonconventional CDK sites when cells progress into mitosis and when mitotic-Cdk1 phosphorylates Swe1 in vitro (Harvey et al., 2005). The docking mechanisms that compensate for the absence of consensus sites are not known for this case. It was recently shown that the Swe1 N-terminus contains an M-CDK specific cyclin-docking motif LxF, which may be one of the factors (Örd et al., 2019b).

In addition to the docking interactions mediated by cyclin subunits, a well-positioned priming phosphorylation site that binds a phospho-adaptor protein Cks1 of the CDK complex (Figure 1A) can facilitate the phosphorylation nonconventional sites. Such a mechanism was demonstrated in the case of phosphorylation of Sic1, the G1 phase inhibitor of CDK complex in budding yeast. The phosphorylation of a critical di-phosphodegron site, the T48 that lacks P+1, was entirely dependent on an intact phospho-binding pocket of Cks1 (Kõivomägi et al., 2011a). A systematic in vitro study addressing the consensus sequence requirements around nonconventional CDK sites showed that these motifs usually contain one or more basic (R/K) amino acids located C-terminally in position +3 or its vicinity. It was concluded that the second crucial element of the consensus motif must still be present (Suzuki et al., 2015). In addition to the +3K/R, a -2 hydrophobic residue was present in nonconventional CDK sites of the critical di-phosphodegrons in Sic1 and Far1 (Kõivomägi et al., 2011a; Faustova et al., 2021), and in several sites discovered in a proteomic screen (Michowski et al., 2020). The -2Pro has been shown to have an enhancing effect on the phosphorylation rate of model substrate sites by budding yeast CDK (Kõivomägi et al., 2011b). Several other studies have demonstrated that a significant fraction of cell cycle regulated phosphorylation is comprised of nonconventional CDK sites enriched in a K in the +3 position (Dephoure et al., 2008). Recently, it was found that a large number of direct Cdk1 substrates in mouse embryonic stem cells were nonconventional CDK sites containing lysines in position +3 and also at positions +2, +4, and +5 (Michowski et al., 2020). A phospho-proteomics approach using fixed TK6 cells and in vitro kinase assays identified a large number of nonconventional CDK sites and came to the same conclusion that for these sites to be phosphorylated, usually, the lysine +3 and Cks1 docking must be present (Al-Rawi et al., 2023).

FIGURE 1:

FIGURE 1:

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The nonconventional CDK sites enhance CDK-signaling specificity in cell cycle regulation. (A) A specificity-filter discrimination between CDK and other proline-directed kinases. The docking mechanisms of the ternary CDK complex provide fixed positioning and defined-binding specificity of docking pockets for cyclin-docking motifs, phospho-priming sites for Cks1 and the kinase active-site (marked with the red line). In this scheme, a nonconventional CDK phosphorylation site is marked as green. The light orange-labeled phosphorylation site is a conventional CDK site with a TP motif that, after being primed by CDK phosphorylation, acts also as a docking site by binding to the phosphate pocket of Cks1. A cyclin-docking motif is depicted as purple rectangle. The optimal multi-docking mechanism enables CDK to phosphorylate the low-specificity nonconventional CDK sites. Contrarily, in case of other proline-dependent kinases, for example the MAP kinases, or other CDK-like kinases that do not contain similar pockets or Cks1-type adaptors, the direct recognition and phosphorylation of the nonconventional sites is less likely. This is because the active-site vicinity lacks sufficient recognition elements and the docking pockets, even if they exist, are not the identical to the ones in CDK complex and thus, would not recognize the CDK-docking motifs in CDK targets. (B and C) Regulatory phosphorylation of nonconventional CDK sites combined with SLiM CDK-docking motifs and optimal/suboptimal distances between them along the disordered regions of phosphorylated targets provide a wide range of options for encoding CDK thresholds, while being shielded from cross-signaling by other proline-directed kinases as shown in (A).

NONCONVENTIONAL CDK SITES AS SPECIFICITY FILTERS TO DISTINGUISH THE CDK SIGNAL FROM OTHER PROLINE-DIRECTED KINASES

Despite the proteome-wide detection of nonconventional CDK consensus sites, very little is known about the role that the nonconventional sites play in cell-cycle regulation. There are only a handful of examples of regulatory function. These include the potential role of nonconventional CDK sites to drive the dissociation of Swe1 from the mitotic cyclin/CDK complex (Harvey et al., 2005). Nonconventional CDK sites were also found to be crucial elements of the di-phosphodegrons responsible for the degradation of Cdk1 inhibitors Sic1 and Far1 (Kõivomägi et al., 2011a; Faustova et al., 2021). An NLS motif matching also the nonconventional CDK site (P-X-S-X-[R/K]5) of the cytokinesis regulator Ect2, and also in conserved linkers (e.g., T-G-E-K-P) of C2H2 zinc finger proteins were shown to be regulated by Cdk1 phosphorylation (Suzuki et al., 2015). In the spindle checkpoint, the binding of the MIS12 complex to CENP-T was shown to be regulated by CENP-T phosphorylation at a nonconventional site S201 by CDK1 (Huisin’T Veld et al., 2016). These established examples of relatively different functions (e.g., regulation of degrons, localization signals, and protein complex formation) indicate that the nonconventional CDK sites could potentially mediate a very wide range of different mechanisms in the cell cycle.

The serine-threonine protein kinase superfamily contains three main subfamilies defined by phosphorylation-site specificity. The two large groups are the basophilic, and proline-directed kinases, whose common-core specificity elements are the -3R, and +1P, respectively. A smaller acidophilic group of kinases recognizes several combinations of acidic amino acids in C-terminal positions from the phosphorylated residue. In addition, there are a few smaller less-defined groups (Johnson et al., 2023). One of the great unsolved questions is how the Ser/Thr-protein kinases within these groups of closely related kinases achieve site-specificities to carry out orthogonal signaling and avoid cross-specificity. For example, how do two different kinases belonging to the same specificity group, whose outputs must lead to different cellular responses, avoid targeting each other’s key phosphorylation target sites despite the overlapping substrate specificity? The problem is especially interesting in higher eukaryotes where, in principle, hundreds of kinases can be expressed simultaneously. A mix of kinases from one specificity group can have a relatively high total concentration. Even if one of these many kinases, using docking interactions, has developed very high specificity towards a consensus phosphorylation site in a target, the numerous other kinases of the same broad-specificity group would gain high net-phosphorylation rate solely via the active site motif specificity and due to the mass action of the high total concentration of the kinase mix. Thus, in case this single specific kinase is switched on to trigger a particular signaling pathway, it would potentially be accompanied by a very strong background signaling from the bulk of other kinases from the same specificity group. There are potentially several possibilities to achieve orthogonal signaling among the group of proline-directed kinases. For example, differential subcellular localization or specific adaptor-protein complexes are one possibility to amplify a particular kinase and it’s signaling pathway.

One example of two different kinases of the same specificity group that signals alternative cell fates can be found in the mating pathway of S. cerevisiae. The CDK target and inhibitor Far1 plays a role in the signal-integration point of the pathway. CDK-mediated phosphorylation of di-phosphodegron in Far1 triggers its degradation and loss of CDK inhibition leading to a CDK-driven cell cycle entry in the G1 phase. Alternatively, the MAP kinase Fus3 that is activated by the pheromone-pathway phosphorylates Far1 at another site to trigger the CDK-inhibiting function of Far1 and the pheromone-arrest of the cells in the G1 phase (Gartner et al., 1998). Both Fus3 and CDK are proline-directed kinases. Here, a nonconventional CDK site in the Far1 di-phosphodegron provides an advantage in achieving the orthogonality (or avoiding cross-specificity) of the two alternative signals. We have shown that a di-phosphodegron in Far1 contains one CDK-consensus site and one nonconventional CDK site (PI87pSPPP91pSLKK; Faustova et al., 2021). For degradation, both sites must be phosphorylated. The phosphorylation of the nonconventional site by CDK is made possible with the help of a highly cyclin-specific docking mechanism that MAPK Fus3 does not possess (Figure 1A). This creates a filter that prevents the leakage of the Fus3 signal during pheromone arrest into the CDK degron and thus protects Far1 from degradation and slippage of the cell into the S-phase (Faustova et al., 2021). A similar di-phosphodegron based filter relying on the nonconventional CDK site T48 was found in Sic1, another key CDK target that controls the G1/S transition in budding yeast (Kõivomägi et al., 2011a). Such a mechanism seems to be conserved in eukaryotes as an analogous di-phosphodegron structure is also present in mammalian Cyclin E at sites LL380TPPQ384SGKK (Hao et al., 2007).

WEAK NONCONVENTIONAL CDK-SITE SPECIFICITY WIDENS THE RANGE OF CDK THRESHOLDS

To assess the wider-physiological importance of nonconventional CDK sites in phospho-regulation, there is still not enough in vivo data about the phosphorylation stoichiometry of these sites. Indirect evidence from studies with Far1 indicated that phosphorylation of nonconventional CDK site S91 was essential for interaction with ubiquitin ligase SCF adaptor Cdc4 in vivo (Faustova et al., 2021). A similar conclusion was reached for nonconventional CDK site T48 in the Sic1 di-phosphodegron (PV45pTPS48pTTK) (Kõivomägi et al., 2011a). However, in the case of Sic1, no considerable accumulation of the multiply phosphorylated species was detected in vivo at G1/S transition using high-resolution Phos-tag SDS–PAGE, which would indicate that phosphorylation of the T48 nonconventional CDK site, as a relatively slow rate-limiting step in the multisite-phosphorylation process, is followed by fast ubiquitination. However, partial phosphorylation of both S91 in Far1 and T48 in Sic1 was detectable in vitro. In the case of S91, phosphorylation was entirely dependent on a highly S-phase CDK-specific docking motif NLxxxL located 40 amino acids C-terminal from S91 site, and in the case of T48 in Sic1, the priming and Cks1 docking site T33 was essential for the electrophoretic-phosphorylation shift in Phos-Tag SDS–PAGE. Although conditions for in vitro phosphorylation may allow modification of sites not phosphorylated in vivo, in the given examples of Sic1 and Far1 the alanine mutations at the nonconventional sites of the degron abolished CDK-dependent degradation, which indicates that in vitro phosphorylation reflects the mechanism in vivo. Thus, as already pointed out above, distant docking via Cks1 and cyclin-docking motifs can facilitate the phosphorylation of nonconventional CDK sites. As these docking mechanisms depend on many factors including the affinity of the docking SLiMs to cyclins and their relative positions or distances from the phosphorylated sites, the phosphorylation rate of nonconventional CDK sites can be distributed over a wide range of efficiencies (Figure 1, B and C). This is in contrast to the optimal phosphorylation motifs, which can abruptly reach the full-phosphorylation stoichiometry already at the G1/S transition in S.cerevisiae (Kõivomägi et al., 2011b). Such a switch to a full-phosphorylation stoichiometry at early-CDK levels does not provide a wide window of accumulating CDK thresholds for ordering cell cycle events from G1/S to mitosis, as the quantitative model of CDK function would predict (Fisher and Nurse, 1996).

Interestingly, the spacing requirements for Cks1-mediated phosphorylation showed a well-defined optimum between the phosphorylated docking site acting as a primer that binds Cks1 and a secondary site to be phosphorylated in the C-terminal direction from the primer. The spacing between the two sites has a minimal exclusion zone of 10 amino acids along the peptide chain as determined using model substrates based on Sic1, followed by a window of about 12–22 amino acids. Within this window, a wide range of Cks1-mediated phosphorylation rates was observed (Kõivomägi et al., 2013). Thus, the distance between the optimal priming site and the suboptimal secondary site can be a tunable parameter that defines the overall rate of multisite phosphorylation.

Many kinases in signal-transduction networks act as binary on-off switches. Contrarily, the CDK, whose role is to coordinate the complex process of the cell cycle via hundreds of targets (Holt et al., 2009; Enserink and Kolodner, 2010), is unique because instead of a simple on-off mechanism, it has to trigger many processes at multiple different activity thresholds. These activity thresholds must be programmed into CDK targets as different specificities via complex docking configurations (Figure 1, B and C). In addition, as explained above, all these nonconventional sites in many CDK targets are shielded from other proline-directed kinases at all stages of the cell cycle, creating orthogonality for CDK signaling and the cell cycle that is unperturbed by the bulk of kinase-signaling noise.

CONSENSUS SITE SWITCHING OR GRADUAL INCREASE OF ACTIVE-SITE SPECIFICITY?

Several studies have demonstrated that cyclins can alter the active-site specificity of Cdk. First, in S.cerevisiae, it was shown that the activity (both in terms of KM and kcat/KM values) of different CDK complexes towards the histone H1 derived minimal model substrate peptide increased gradually in the order of their appearance of the cyclin in the cell cycle (Loog and Morgan, 2005; Kõivomägi et al., 2011b). Similar results were more recently obtained with mammalian CDK complexes (Topacio et al., 2019). The precise mechanistic cause for such gradual change is not known, but different cyclins can likely introduce different conformational effects on the CDK-kinase subunit and its active-site cleft. Thus, early CDK complexes are kinases with relatively weak activity, exhibiting relatively low kcat/KM values for optimal short peptide substrates that are recognized only by the active site of CDK. This disadvantage at the active-site level for early CDK complexes can be compensated by cyclin-specific docking interaction via SLiMs in CDK substrates regulating the early phases of the cell cycle (Schulman et al., 1998; Wilmes et al., 2004; Loog and Morgan, 2005; Bhaduri and Pryciak, 2011; Örd and Loog, 2019).

Interestingly, a recent proteomic study revealed that nonconventional sites were more associated with mammalian Cyclin A signaling, while sites detected for Cyclin B/CDK1 phosphorylation were closer to a conventional +1P profile (Al-Rawi et al., 2023). The term “consensus-site switching” was used by the authors to describe this apparent change in phosphorylation-site specificity during the cell cycle progression as Cyclin A expression precedes that of Cyclin B. This is an important observation but does not necessarily indicate that a consensus site switching occurs during the sequential appearance of Cyclin A and B in the course of the cell cycle. Rather, the result could simply reflect the lower activity of Cyclin A/CDK towards the consensus site compared with Cyclin B complex (Topacio et al., 2019). For Cyclin A, the absence of the full consensus sites is likely compensated by extra-docking mechanisms. In this case, the sites with only partially consensus motif - nonconventional CDK sites with no +1P but with +3K/R, or even random S or T residues may start to become phosphorylated. Thus, the observation could just be the manifestation of the gradually increasing intrinsic activity of CDK complexes and the compensation of the weak specificity of early complexes by a specific-docking mechanism, which prompts also wide-collateral phosphorylation of partial-consensus sites. The later mitotic Cyclin B/CDK complex with high-intrinsic specificity relies mostly on specific low-micromolar KM values for phosphorylation consensus sites and uses docking mechanisms less frequently (Örd et al., 2019b).

Indeed, some level of consensus site switching has been previously observed. The G1 cyclins in S.cerevisiae introduce small changes in the conventional active site specificity of Cdk1. The Cln1/2-Cdk1 complexes have an additional specificity determinant +2K/R that alone provides similar specificity as the conventional +3K/R (Kõivomägi et al., 2011b). The Clb-Cdk1s are insensitive to the positive charge in this position. Recently it was reported that the G1 cyclin Cln3 could use an additional specificity determinant -4F/Y (Kõivomägi et al., 2021).

PERSPECTIVES

Only a handful of regulatory examples involving nonconventional CDK sites have been defined so far with sufficient mechanistic detail. In a few cases, the precise docking requirements for the phosphorylation of such sites were demonstrated, including S-CDK specific cyclin docking (Kõivomägi et al., 2011a; Faustova et al., 2021) or Cks1-dependent docking (Kõivomägi et al., 2013; McGrath et al., 2013; Örd et al., 2019a). It would also be interesting to test whether the recently discovered phosphate-docking pocket, conserved in B-type cyclins, would mediate the phosphorylation of nonconventional sites via priming phosphorylation, similar to the Cks1-dependent mechanism, and whether the phosphorylated nonconventional sites could form a docking motif for the phospho-pocket (Yu et al., 2021; Asfaha et al., 2022). This conserved phosphate-binding pocket in cyclin B1 is a recent discovery that emerged from the structure of human separase in complex with cyclin B1-CDK1-CKS1 (Yu et al., 2021). Recently, it was shown that the pocket can promote secondary phosphorylation in cooperation with Cks1. The Clb2-Cdk1-Cks1 was used in vitro kinase assay with budding-yeast transcriptional regulator Ndd1 as a substrate (Asfaha et al., 2022). Another interesting element to study would be the role of phosphatases. For example, how a +1P-specific phosphatase such as Cdc14 acting in mitotic exit would be able to control the switches mediated by nonconventional CDK sites? The examples of the di-phosphodegrons already provide answers: these degrons contain one conventional and one nonconventional CDK site that acts in an AND-gate-like manner. The condition for degradation is that both sites must be phosphorylated. Thus, even if the Cdc14 can catalyse the removal of the phosphate from only one of the two sites of the degron - the conventional site with +1P, it would be sufficient to switch off the degradation.

It is highly likely that a large fraction of crucial-CDK signaling is transmitted via nonconventional CDK sites. However, it remains to be determined how large this fraction is, and what are the mechanistic details and the emerging properties of such signaling. Furthermore, phosphorylation of nonconventional sites, driven by similar mechanisms, will likely apply in many other signaling networks. For example, proteomic studies of MAP kinases Fus3 phosphorylation revealed many phosphorylated nonconventional sites (Repetto et al., 2018).

In conclusion, as CDK function in the cell cycle involves multisite phosphorylation of its targets, studying only conventional CDK sites is not sufficient to understand the mechanism of CDK-regulated processes. The described nonconventional sites together with cyclin-docking motifs and Cks1-binding priming phosphorylation sites must be taken into account.

Abbreviations used:

CDK

cyclin-dependent kinase

SLiMs

Short Linear Motifs.

Footnotes

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