The Eukaryotic Linear Motif resource for
Functional Sites in Proteins
Accession:
Functional site class:
Cdc14 phosphatase dephosphorylation site
Functional site description:
Progression through the cell cycle is driven by sequentially changing sets of substrates getting phosphorylated by Cdk(s) coupled to cyclin activators with distinct substrate specificities. In yeast, Cdc14 is a widely conserved, dimeric, cell-cycle regulated phosphatase that is a key regulator of mitotic exit events by dephosphorylating a subset of Cdk1 substrates. It is inhibited by Net1 during most of the cell cycle and gets only activated during mitotic exit, leading to its nuclear translocation and the sequential dephosphorylation of its targets. Although Cdc14 homologues show differences in the timing, targets and functional readout of their phosphatase activities, their target specificity for (S)PxK/r phosphosites is mostly conserved. Interestingly, docking of the non-catalytic, N-terminal domain of budding yeast Cdc14 to a PxL motif on substrates can facilitate the recognition and dephosphorylation even of substrates carrying suboptimal phosphosites, a mechanism yet to be validated in other organisms.
ELM Description:
The optimal Cdc14 target recognition consensus motif (S)P.[Kr] was derived from mutational analyses combined with in vitro phosphopeptide dephosphorylation assays (Bremmer,2012). The results suggested that phosphothreonines are very weak substrates of Cdc14, proline is absolutely required in the +2 position and that a positive residue is strongly favoured in the +4 position, with lysine being more favourable for efficient catalysis than arginine. Additional positive charges in the +3, +5 or +6 positions confer slight improvements in catalytic efficiency (Bremmer,2012), but they are not strictly required based on the experimentally validated instances. Although the human Cdc14 homologues, CDC14A and CDC14B and the fission yeast Clp1 showed similar residue preferences in peptide dephosphorylation assays, and the residues contributing to the active site are highly conserved (Bremmer,2012), phosphosites of their experimentally validated substrates suggest that they have a more relaxed recognition pattern, wherein the +4 position does not require a positively charged residue.
The structure of the complex between a phosphatase-dead mutant of Cdc14 and an S-phosphorylated Swi6p peptide provides information on the catalytic mechanism of Cdc14 (5XW5). A structure of human CDC14B with a substrate peptide is also available, but unfortunately the peptide is too short to contain the positive residues in the positions following the (S)P (1OHE; Gray,2003). Phosphothreonines are poor Cdc14 substrates, mainly due to a steric clash with the methyl group of a conserved alanine residue contributing to the phosphatase active site (Bremmer,2012; Eissler,2014).
Results of high-throughput phosphoproteomics studies (Eissler,2014; Powers,2017) and peptide docking simulations (Eissler,2014) also confirmed the (S)P.[Kr] motif.
Pattern: (S)P.[KR]
Pattern Probability: 0.0012270
Present in taxon: Eukaryota
Interaction Domain:
DSPc (PF00782) Dual specificity phosphatase, catalytic domain (Stochiometry: 1 : 1)
o See 48 Instances for MOD_CDC14_SPxK_1
o Abstract
Progression through the cell cycle is driven by sequentially changing sets of substrate proteins getting phosphorylated by Cdk(s). This is ensured by sequentially expressed cyclin activators that confer distinct substrate specificities on Cdk(s) in different phases of the cycle. During the cell cycle, Cdk activity is modulated by opposing phosphatase activities. In budding yeast, Cdc14 acts as one of the antagonists of Cdk activity regulating a well-defined subset of Cdk substrates mainly participating in late mitotic events such as chromosome segregation, spindle disassembly, DNA damage response and cytokinesis (Bremmer,2012; Eissler,2014; Kao,2014; Powers,2017; Mocciaro,2010; Villoria,2017; Manzano-Lopez,2020). Although Cdc14 homologues have been identified in a wide range of eukaryotes and their target specificities are mostly conserved (Bremmer,2012; Li,2015; Vazquez-Novelle,2005), the key role played in mitotic exit is yet only confirmed in budding yeasts. For instance, the fission yeast Cdc14 homologue, Cdc14-like phosphatase 1 (Clp1; also known as Flp1; Q9P7H1), contributes to the control of cytokinesis, but is not required for other aspects of mitotic exit or Cdk1 inactivation (Trautmann,2004). The Caenorhabditis elegans Cdc14 homologue, cdc-14 (P81299), does not function in mitosis, rather it is crucial for G(1)/S regulation to establish developmental cell-cycle quiescence (Saito,2004). Among the vertebrate homologues, CDC14B (O60729) is functionally related to yeast Cdc14 and plays major roles in functions such as the G2/M DNA damage checkpoint, DNA repair and centrosome duplication, while CDC14A (Q9UNH5) is involved in centrosome separation and cytokinesis, suggesting that the functions of CDC14 phosphatases have been partially rewired during eukaryotic evolution but preserve important hallmarks (Mocciaro,2010; Powers,2017).
The yeast Cdc14 protein belongs to the dual-specificity phosphatase family (PF00782). Cdc14 is sequestered in the nucleolus for most of the cell cycle by the nucleolar proteins Net1 and Tof2, and is only released into the nucleoplasm and cytosol during anaphase (Visintin,1999; Shou,1999). In anaphase, the so called Fourteen Early Anaphase Release (FEAR) network (including Cdc5, Esp1, and Slk19), and the mitotic exit network (MEN; including the Dbf2-Mob1 complex) co-ordinately trigger the release of Cdc14 from the nucleolus (Shou,1999). Subsequently, Cdc14 performs the sequential dephosphorylation of its targets (Bouchoux,2011). Both the molecular mechanism of (in)activation and the timing of activity during the cell cycle show remarkable differences between Cdc14 homologues (Powers,2017; Manzano-Lopez,2020).
In vitro, Cdc14 strongly favours dephosphorylation of phosphoserines followed by a proline, with an additional positively charged residue (preferentially lysine) in the +4 position ((S)PxK/r) which, maybe not surprisingly, conforms to a restricted version of the Cdk consensus phosphorylation motif (Bremmer,2012; MOD_CDK_SPxK_1). Phosphothreonines are poor Cdc14 substrates, due to a steric clash with the methyl group of a conserved alanine residue of the phosphatase active site (Bremmer,2012; Eissler,2014). In vitro phosphatase assays using wildtype or mutated phosphorylated target peptides clearly indicated that lysine in the +3 position enables higher catalytic efficiency than arginine (Bremmer,2012; Eissler,2014). Also, additional positively charged residues in the +3, +5 or +6 positions of the phosphorylated target motifs further enhance the catalytic efficiency of yeast Cdc14 (Bremmer,2012). Based on the experimentally validated targets of Cdc14, lysine over arginine in the +4 position and the additional positively charged residues in the surrounding positions are not required for dephosphorylation by Cdc14, however, they could influence in vivo catalytic efficiency and therefore may determine the order of the different substrates getting dephosphorylated after the activation of the phosphatase.
Due to the low affinity and transient nature of the kinase/phosphatase-substrate interactions, the substrates are generally difficult to identify. The substrates of Cdc14 have been best characterized in budding yeast. The phosphatase dead mutants of yeast Cdc14 (C283S or D253A) have been declared as promising “substrate traps” and they were used to identify the potential in vivo substrates of Cdc14 in pull down and co-immunoprecipitation experiments (Bloom,2011). These substrate trap experiments could only identify a small subset of the potential Cdc14 targets detected in high-throughput phosphoproteomic screens (Bloom,2011; Eissler,2014; Powers,2017). Although many substrates of yeast Cdc14 have been validated in low-throughput experiments, the changes in the phosphorylation states of full proteins encompassing several phosphosites were mainly demonstrated by mass-dependent changes in migration within gels, therefore the dephosphorylation of individual phosphosites by Cdc14 has rarely been addressed. Nevertheless, analysis of the peptide hits of large-scale phosphoproteomic analyses of potential Cdc14 targets have confirmed the in vivo relevance of the (S)PxK/r motif (Eissler,2014; Powers,2017).
Fission yeast and human Cdc14 homologues showed similar residue preferences to budding yeast Cdc14 in peptide dephosphorylation assays, and the residues contributing to the active site are highly conserved, therefore the target motif was declared to be widely conserved among eukaryotes (Bremmer,2012).
Also, the structure of the complex between a phosphatase dead mutant of Cdc14 and an S-phosphorylated Swi6p peptide provides information on the catalytic mechanism of Cdc14 (5XW5). Furthermore, a complex structure of human CDC14B and a short substrate peptide is also available, even though the substrate peptide is too short for assessing the preference for positive residues in the positions following the (S)P (1OHE; Gray,2003).
Yeast Cdc14 forms a dimer, and dimerization is essential for its catalytic efficiency (5XW4; Kobayashi,2017). Furthermore, the non-catalytic, N-terminal domain of yeast Cdc14 (PF14671) has a binding pocket that recognizes the PxL docking motif (DOC_CDC14_PxL_1) on its substrates (6G86; 6G85), enhancing target recognition and dephosphorylation of both optimal and suboptimal targets (Kataria,2018). Although the hydrophobic pocket of Cdc14 that interacts with the PxL motif is conserved in human Cdc14 orthologues (Kataria,2018), its functional relevance is yet to be validated outside the phylogenetic group of budding yeasts.
o 10 selected references:

o 7 GO-Terms:

o 48 Instances for MOD_CDC14_SPxK_1
(click table headers for sorting; Notes column: =Number of Switches, =Number of Interactions)
Acc., Gene-, NameStartEndSubsequenceLogic#Ev.OrganismNotes
Q12369 SFI1
SFI1_YEAST
892 895 TRGKPLRYSPRRTTRNMPSK TP 2 Saccharomyces cerevisiae S288c
1 
Q12369 SFI1
SFI1_YEAST
882 885 ESTTGLNGSPTRGKPLRYSP TP 2 Saccharomyces cerevisiae S288c
1 
Q12369 SFI1
SFI1_YEAST
855 858 SRARRAIPSPVKSSSVLDST TP 2 Saccharomyces cerevisiae S288c
1 
Q12369 SFI1
SFI1_YEAST
801 804 LWKNRSDSSPKRRKDFNLKH TP 2 Saccharomyces cerevisiae S288c
1 
Q9HDY1 ase1
ASE1_SCHPO
693 696 MKISPIRASPVKTIPSSPSP TP 1 Schizosaccharomyces pombe 972h-
1 
Q9HDY1 ase1
ASE1_SCHPO
688 691 LPFSPMKISPIRASPVKTIP TP 1 Schizosaccharomyces pombe 972h-
1 
Q9HDY1 ase1
ASE1_SCHPO
683 686 NSSQNLPFSPMKISPIRASP TP 1 Schizosaccharomyces pombe 972h-
1 
Q9HDY1 ase1
ASE1_SCHPO
640 643 ASSKNVPLSPPKQRVVNEHA TP 1 Schizosaccharomyces pombe 972h-
1 
Q1MTQ7 klp9
KLP9_SCHPO
611 614 VVSPIKPLSPSRRPPLTSLY TP 1 Schizosaccharomyces pombe 972h-
1 
Q1MTQ7 klp9
KLP9_SCHPO
605 608 PSPKKKVVSPIKPLSPSRRP TP 1 Schizosaccharomyces pombe 972h-
1 
Q1MTQ7 klp9
KLP9_SCHPO
598 601 DEENVPSPSPKKKVVSPIKP TP 1 Schizosaccharomyces pombe 972h-
1 
P32380 SPC110
SP110_YEAST
36 39 NTTQTQVVSPTKVPNANNGD TP 3 Saccharomyces cerevisiae S288c
1 
P38634 SIC1
SIC1_YEAST
76 79 TSPFNGLTSPQRSPFPKSSV TP 3 Saccharomyces cerevisiae S288c
1 
P08153 SWI5
SWI5_YEAST
664 667 DGTSSVSSSPIKENINKDHN TP 3 Saccharomyces cerevisiae (Baker"s yeast)
1 
P08153 SWI5
SWI5_YEAST
646 649 YENVVIKRSPRKRGRPRKDG TP 3 Saccharomyces cerevisiae (Baker"s yeast)
1 
P08153 SWI5
SWI5_YEAST
522 525 EINTYTTNSPSKITRKLTTL TP 3 Saccharomyces cerevisiae (Baker"s yeast)
1 
P53197 CDH1
CDH1_YEAST
436 439 AAVKAMAWSPHKRGVLATGG TP 3 Saccharomyces cerevisiae S288c
1 
P53197 CDH1
CDH1_YEAST
239 242 PDSKQLLLSPGKQFRQIAKV TP 7 Saccharomyces cerevisiae S288c
4 
P53197 CDH1
CDH1_YEAST
227 230 QSQFFDSMSPVRPDSKQLLL TP 3 Saccharomyces cerevisiae S288c
1 
P53197 CDH1
CDH1_YEAST
42 45 SSSASLLSSPSRRSRPSTVY TP 7 Saccharomyces cerevisiae S288c
3 
P53197 CDH1
CDH1_YEAST
16 19 FMNNTPSSSPLKGSESKRVS TP 5 Saccharomyces cerevisiae S288c
1 
Q08981 ACM1
ACM1_YEAST
3 6 MISPSKKRTILSSKNINQKP TP 7 Saccharomyces cerevisiae S288c
4 
Q12280 IQG1
IQG1_YEAST
404 407 FKYSPSHYSPMRRERMTEEQ TP 3 Saccharomyces cerevisiae S288c
1 
Q12280 IQG1
IQG1_YEAST
354 357 GPTPSLEYSPIKNKSLSYYS TP 3 Saccharomyces cerevisiae S288c
1 
Q12280 IQG1
IQG1_YEAST
7 10 MTAYSGSPSKPGNNNSYLNR TP 3 Saccharomyces cerevisiae S288c
1 
Q08981 ACM1
ACM1_YEAST
31 34 VVKGNELRSPSKRRSQIDTD TP 10 Saccharomyces cerevisiae S288c
5 
Q9UHB6 LIMA1
LIMA1_HUMAN
374 377 RASSLSESSPPKAMKKFQAP TP 4 Homo sapiens (Human)
1 
P09959 SWI6
SWI6_YEAST
160 163 SDAHRELGSPLKKLKIDTSV TP 7 Saccharomyces cerevisiae S288c
1 
Q08981 ACM1
ACM1_YEAST
48 51 DTDYALRRSPIKTIQISKAA TP 6 Saccharomyces cerevisiae S288c
1 
P40316 PDS1
SECU_YEAST
71 74 YIQGGKEVSPTKRLHTHAQQ TP 3 Saccharomyces cerevisiae S288c
1 
P14180 CHS2
CHS2_YEAST
100 103 RYAANLQESPKRAGEAVIHL TP 6 Saccharomyces cerevisiae S288c
1 
P14180 CHS2
CHS2_YEAST
69 72 PSRAALRYSPDRRHRTQFYR TP 6 Saccharomyces cerevisiae S288c
1 
P14180 CHS2
CHS2_YEAST
60 63 NVFQGLPASPSRAALRYSPD TP 6 Saccharomyces cerevisiae S288c
1 
P14180 CHS2
CHS2_YEAST
14 17 FMVEPSNGSPNRRGASNLSK TP 6 Saccharomyces cerevisiae S288c
1 
P40028 YEN1
YEN1_YEAST
679 682 VWKDVIEISPIKKSRTTNAE TP 5 Saccharomyces cerevisiae S288c
1 
P40028 YEN1
YEN1_YEAST
655 658 NSQSVLDSSPGKRIRDLTQD TP 5 Saccharomyces cerevisiae S288c
1 
P40028 YEN1
YEN1_YEAST
583 586 DEFLRKHTSPIKSIGKVGES TP 5 Saccharomyces cerevisiae S288c
1 
P40028 YEN1
YEN1_YEAST
500 503 PNIPISSQSPLKRSNSPSRS TP 5 Saccharomyces cerevisiae S288c
1 
P40028 YEN1
YEN1_YEAST
67 70 ARSRSRSRSPTRSPRDSDID TP 5 Saccharomyces cerevisiae S288c
1 
P25558 BUD3
BUD3_YEAST
1549 1552 QKDEPIWVSPSKIDFADLSR TP 4 Saccharomyces cerevisiae S288c
1 
Q12267 SMC4
SMC4_YEAST
128 131 RRLELLQLSPVKNSRVELQK TP 4 Saccharomyces cerevisiae S288c
1 
P09119 CDC6
CDC6_YEAST
372 375 QVPLTPTTSPVKKSYPEPQG TP 2 Saccharomyces cerevisiae (Baker"s yeast)
1 
P09119 CDC6
CDC6_YEAST
43 46 FTDVTPESSPEKLQFGSQSI TP 2 Saccharomyces cerevisiae (Baker"s yeast)
1 
Q03898 FIN1
FIN1_YEAST
148 151 ARFKNGLMSPERIQQQQQQH TP 2 Saccharomyces cerevisiae S288c
1 
Q03898 FIN1
FIN1_YEAST
117 120 ITNIIFPTSPTKLTFSNENK TP 2 Saccharomyces cerevisiae S288c
1 
Q03898 FIN1
FIN1_YEAST
54 57 FLKPPMRISPNKTDGMKHSI TP 2 Saccharomyces cerevisiae S288c
1 
Q03898 FIN1
FIN1_YEAST
36 39 NVFVRLSMSPLRTTSQKEFL TP 2 Saccharomyces cerevisiae S288c
1 
Q08981 ACM1
ACM1_YEAST
102 105 PSQVKENLSPAKICPYERAF TP 2 Saccharomyces cerevisiae S288c
1 
Please cite: ELM — the eukaryotic linear motif resource in 2020. (PMID:31680160)

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