Two maize Kip-related proteins differentially interact with, inhibit and are phosphorylated by cyclin D–cyclin-dependent kinase complexes

Highlight Maize Kip-related proteins can be differentially phosphorylated by different cyclin D–cyclin-dependent kinase complexes and this influences their performance as cyclin-dependent kinase inhibitors.


Introduction
The life cycle in plants is highly coordinated by regulation of cell cycle events and developmental programs. Control of cell cycle progression in eukaryotes is dependent on a conserved molecular machinery consisting of a number of protein kinases known as cyclin-dependent kinases (CDKs) that are activated after binding to regulatory proteins named cyclins (Cyc) (Vidal and Koff, 2000;Vandepoele et al., 2002).
Several additional factors regulate kinase activity in these cyclin-CDK complexes: stimulation of CDK activity after phosphorylation in Thr 161 by a CDK activating kinase (CAK; Murray, 2004); inhibition by phosphorylation of residue Tyr 15 by the WEE1 kinase (Nakagami et al., 2002;Dewitte and Murray, 2003); and inhibition by binding to proteins known as inhibitors of CDKs (ICKs) that arrest cell cycle progression in response to internal or external cues (Verkest et al., 2005).
In mammals, the family of Cip/Kip proteins inhibits a wide range of cyclin-CDK complexes in the G1-S transition (Stals & Inzé, 2001;Leibovitch et al., 2003) and these proteins apparently also function for assembly and stabilization of cyclin-CDK complexes, or even for transporting complexes into the nuclei, processes in which Cip/Kip proteins can also be phosphorylated by the same kinase complexes (McConnell et al., 1999). Thus, cell cycle regulation by these proteins requires physical contact with CDKs (Leibovitch et al., 2003;Lacy et al., 2004) with low concentrations required for complex stabilization or higher concentrations for inhibition of kinase activity (Zhou et al., 2003a, b).
In plants, two families of related CDK inhibitors have been described. The first is the Arabidopsis plant-specific SIAMESE/SIAMESE RELATED (SIM/SMR) proteins (Churchman et al., 2006;Peres et al., 2007), which are involved in the regulation of endoreduplication events (Kumar et al., 2015), with some members regulating the innate immune response during interaction with pathogens (Hamdoun et al., 2016) and others regulating cell cycle checkpoints in response to DNA damage induced by oxidative stress (reactive oxygen species) (Yi et al., 2014). The second is the inhibitor of CDK (ICK)/Kip-related protein (KRP) family, which shows a limited similarity to mammalian p27 Kip1 protein (Wang et al., 1997;De Veylder et al., 2001). This is composed of seven subgroups, originally identified in Arabidopsis . By sequence alignment and phylogenetic trees, ICK/KRP proteins have been classified into three groups: class A exclusively for dicotyledonous plants, class B only for monocotyledonous plants, and class C present in both monoand dicotyledonous plants (Torres Acosta et al., 2011).
There are nine motifs that are conserved among ICK/ KRP proteins (Torres Acosta et al., 2011). Motifs 1 and 2 are essential for their inhibitory activity since motif 1 is required for CDK binding and kinase inhibition (Zhou et al., 2003a), whereas motif 2 is important for interaction with cyclin D (Wang et al., 1998); motif 7 confers nuclear localization to ICK/KRPs (Jakoby et al., 2006;Bird et al., 2007). Little is known about the function of the other motifs.
Overexpression of ICK/KRP genes in plants shows some common phenotypes that include reduction in plant size, serrated leaves, reduction in cell number and cell elongation (De Veylder et al., 2001;Jasinski et al., 2002bJasinski et al., , 2003Barrôco et al., 2006;Wang et al., 2007), and in some cases infertility (Zhou et al., 2002).
Different plant tissues express various members of the ICK/KRP family, although with varied levels, suggesting specific regulatory mechanisms (Wang et al., 1998;De Veylder et al., 2001, Jasinski et al., 2002aOrmenese et al., 2004). Also, the differential expression of different ICK/KRPs during the cell cycle suggests phase-specific regulation (Menges et al., 2005), probably indicating a non-redundant role of the different ICK/KRPs during cell proliferation and differentiation (Torres Acosta et al., 2011).
All Arabidopsis ICK/KRPs and at least one from tobacco interact with cyclins D (De Veylder et al., 2001;Jasinski et al., 2002b;Zhou et al., 2002) and inhibition of the associated kinase activity reduces cell cycle progression and DNA content, depending on KRP concentration (Verkest et al., 2005). Interaction of KRPs with other Cyc-CDK complexes has also been demonstrated, since in alfalfa ICK/KRPs bind CDKB2;1 (Pettkó-Szandtner et al., 2006) and in maize bind CycA (Coelho et al., 2005).
Little is known about ICK/KRP regulation in plants at the protein level; Zeama;KRP2 and Arath;KRP2 seem to be regulated by proteolysis during endosperm development (Zea mays; Coelho et al., 2005) or leaf development (Arabidopsis; Verkest et al., 2005). In Arabidopsis KRP2 is phosphorylated by CDKA1 or CDKB1;1 kinase complexes and thus targeted for proteolysis (Verkest et al., 2005).
DNA endoreduplication is observed in weak overexpressing ICK1/KRP1 or ICK2/KRP2 transgenic Arabidopsis plants resulting in higher ploidy levels (Verkest et al., 2005;Weinl et al., 2005); with high overexpression, cell cycle progression is inhibited and ploidy is strongly reduced as well as cell number. Whereas single Arath ICK/KRP mutants show no evident phenotypes, multiple mutants show diverse morphological changes such as longer cotyledons, leaves, petals and seeds compared with wild type plants. These multiple mutants show more cells but with reduced size in every organ examined (Cheng et al., 2013).
During maize germination, the advance of cell cycle events is required for germination completion and establishment of a plantlet (Vázquez-Ramos and Sánchez, 2003). Our group has demonstrated the presence of different cyclin D-CDK complexes that show differential associated kinase activity during the germination process (Godínez-Palma et al., 2013); moreover, a maize KRP protein (classified as KRP4;2 in this paper) was shown to inhibit kinase activity in CycDassociated CDKs (De Jesús Juárez et al., 2008;Lara-Núñez et al., 2008).
In this work we have studied the expression of maize ICK/ KRPs in different tissues and during seed germination and determined the inhibitory activity that two members of this family (KRP1;1 and KRP4;2) have over different CycD-CDK complexes. We have found that both KRPs are phosphorylated by different CycD-CDK complexes and moreover, that KRP4;2, previously phosphorylated by either CycD2;2-CDKA or CycD6;1-CDKA recombinant complexes, exhibits different inhibitory activity on CycD-CDK complexes immunoprecipitated from cells. We discuss the physiological significance of these results.
For the alignment of maize, rice and Arabidopsis ICK/KRP sequences, Clustal W Multiple Alignment (Thompson and French, 1994) in BioEdit was used; results were kept in FASTA format and were imported to MEGA5.0 (http://en.bio-soft.net/tree/MEGA. html) in order to be analysed with the neighbor-joining method. The phylogenetic tree was built with the FigTree v1.3.1 program (http:// tree.bio.ed.ac.uk/software/figtree/). The identity matrix of maize and rice sequences was obtained by using BioEdit and then transformed to percentages.

RNA extraction
For ICK/KRP gene expression during maize germination, five imbibition times were chosen (0, 6, 12, 18 and 24 h). Chalqueño maize seeds were used (harvest 2013) and were disinfected with sodium hypochlorite (3%), rinsing with deionized water. Then seeds received a 5% solution of Sin-Bac® (bromo-chloro dimethyl hidantoin). Seeds were placed between paper towels with deionized water and after each imbibition time embryo axes were dissected. Different plant tissues were used, leaves (base and tips), roots and coleoptile; for this, plantlets were kept in the dark for 3 days at 25 °C and then changed to a photoperiod regime (16 h light/8 h dark) at 25 °C. Plantlet tissues were obtained after 14 d post-germination. Leaf tissue selection was as reported by Li et al. (2010), and the second leaf was selected because it complied with the recommended dimensions (~14 cm long). For primary root tissues, the first 2 mm was used and for coleoptile, the tissue surrounding the stem was recovered and used.
The corresponding tissues (100 mg) were frozen in liquid nitrogen; tissues were homogenized and 1 ml of TRIzol Reagent (Invitrogen) was finally added. Extraction was as indicated by the provider, but extracting twice with chloroform. Samples were quantified and monitored by means of agarose gels (1%).
cDNA preparation RNA was calibrated loading 400 ng and the concentration was adjusted after densitometric analysis. Contaminant DNA was removed by incubation with 1 U of RQ1 DNase (Promega) per microgram of RNA, 2 µl 10× Reaction Buffer, in a final volume of 20 µl, at 37 °C for 30 min; then, 2 µl of RQ1 DNase Stop Solution (Promega) was added and incubated 10 min at 60 °C to inactivate the enzyme. cDNA was synthesized by adding 1 µg of RNA using the Improm-IITM Reverse Transcription System kit (Promega) following the instructions of the provider.

PCR amplification
Specific primers were designed for the different genes and the PCR reaction was performed using the JumpStart TM Taq ReadyMix TM enzyme (Sigma-Aldrich). Amplification conditions are shown in Supplementary Table S3 at JXB online. cDNA (1 μl) synthesized from total RNA was used as template in a reaction containing 10 μM of each dNTP, 5× Q5 Reaction Buffer and Q5 High-Fidelity DNA polymerase (0.5 U). For cloning of KRPs, the PCR product was monitored in agarose gels (1%); the band corresponding to the cDNA of each ICK/KRP was cut and purified using the GenElute TM Gel Extraction Kit (Sigma-Aldrich), following the provider's instructions.

Recombinant proteins
Purified cDNAs corresponding to KRP4;2 and KRP1;1 were ligated to pGEM-T-easy vector (Promega) using the procedure recommended by the manufacturer. Competent E. coli, XL1-blue cells were transformed and positive cells were grown in LB medium at 37 °C for 1 h; then cells were centrifuged, the pellet was resuspended (LB-ampicillin) and cells were grown in agar LB-ampicillin supplemented with X-Gal and isopropyl β-D-1-thiogalactopyranoside (IPTG; 0.5 mM).
Selected colonies were grown in LB-ampicillin, cell extracts were prepared and the plasmid was isolated using the Zyppy TM Plasmid Miniprep kit (Biasys). Plasmids cut by EcoRI were confirmed by double restriction with NotI (NEB) and BamHI (Invitrogen) enzymes and PCR. After double restriction, the resulting fragments were separated by agarose gel electrophoresis (1.2%) and bands were cut and purified. The recovered fragments were ligated to the pPROEX HTb vector (Invitrogen) and these plasmids were transformed into competent E. coli XL1-blue cells. Plasmids were recovered and inserts checked again by double restriction and by sequencing. Nucleotide and amino acid sequences were analysed with Translate from ExPASY Bioinformatics Resource Portal (http://expasy.org/tools/) and multiple alignment using Clustal W in BioEdit, respectively.
To purify recombinant His-KRP1;1 and His-KRP4;2 proteins, LB medium plus IPTG was used (100 ml) incubating at 37 °C for 3 h. Bacterial extracts were spun at 2741 g for 15 min, and the pellet was resuspended in 5 ml of buffer B (100 mM NaH 2 PO 4 , 10 mM Tris-Cl and 8 M urea, pH 8.0) and incubated for 1 h at room temperature. The lysate was centrifuged at 11 447 g for 20 min at room temperature. The supernatant was added to Ni-NTA agarose resin (Qiagen) for 1 h at room temperature with shaking and washed twice with 4 ml buffer C (100 mM NaH 2 PO 4 , 10 mM Tris-Cl and 8 M urea, pH 6.3). Recombinant proteins were eluted with buffer E (100 mM NaH 2 PO 4 , 10 mM Tris-Cl and 8 M urea, pH 4.5).
In vitro interaction of recombinant proteins E. coli strains overexpressing the different recombinant proteins obtained in our lab (GST-CDKA, His-KRP4;2, GST-CycD6;1, PeX-CycD6;1 and CycD2;2-MBP), were grown in LB medium with the corresponding antibiotics (see Supplementary Table S4). Cultures (5 ml) were incubated for 16-18 h at 37 °C with shaking and then were added to 100 ml fresh LB medium and incubated at 37 °C with the corresponding antibiotics until OD 600nm around 0.5-0.6 was reached. IPTG was then added to induce expression of the recombinant proteins. After induction, growth media were transferred to 250 ml centrifuge bottles, centrifuged at 2741 g and the pellet was resuspended in 10 ml of PBS 1× + 0.05% Tween 20. A tablet of Complete Protease Inhibitors was then added and cells were lysed with lysozyme (Sigma-Aldrich), finally adding 6 mM MgCl 2 , 1 mM EDTA and Benzonase (Sigma-Aldrich, 1.5 U ml -1 ), incubating for 30 min at 37 °C.

Purification of p-His-KRP4;2
After CycD-CDK phosphorylation of KRP4;2, proteins were heat-denatured, loaded to the PhosphoProtein Purification Kit column (Qiagen) and incubated for 30 min at 4 °C in the purification column. Then proteins were eluted, collected, and this procedure repeated five times. In every purification, the amount of resin used was calculated to purify around 25 µg of recombinant p-His-KRP4 protein; column washing conditions were as indicated by the manufacturer, collecting 500 µl fractions. The final elutions were desalted and then were pooled and concentrated by means of Amicon Ultra and protein was quantified. Identification of the phosphorylated protein was achieved by western blot, using the anti-pT-P/pS-P antibody (1:1000 dilution) (see Supplementary Fig.  S8A, B). Quantification of p-KRP4;2 protein was by the method of Bradford (1976).

In silico characterization and nomenclature of maize ICK/KRPs
The search for maize genes (Ensembl plants; http://plants. ensembl.org; Kersey et al., 2016) orthologous to the ICK/ KRPs present in rice (Oryza sativa) and Arabidopsis revealed the existence of nine genes conserving the carboxyl-terminal region required for inhibition of kinase activity in CDK proteins (Zhou et al., 2003a, b; accession numbers are shown in Supplementary Tables S1 and S2). Of these, four corresponded to genes previously reported: Zeama;ICK1, Zeama;ICK2 (Coelho et al., 2005), Zeama;ICK3 and Zeama;ICK4 (Torres Acosta et al, 2011), and two seem to be duplications of the same gene (now named KRP1;2a and KRP1;2b), since they have identical coding sequence and only differ in the 5′ untranslated region (see Supplementary Fig. S1A), being both in different loci (see Supplementary Table S2).
Plant ICK/KRPs (including maize genes) have been classified according to their chronological appearance; however, in this work we propose a nomenclature for maize genes that is based on percentage of identity and phylogenetic comparison particularly with rice genes (Fig. 1A and Supplementary  Fig. S1B). Thus, four sequences seem to be more related to Orysa;KRP1, one to Orysa;KRP3, two to Orysa;KRP4 and two to Orysa;KRP5; there were no sequences related to Orysa;KRP2 and Orysa;KRP6 ( Fig. 1A and Supplementary Fig. S1B).
Protein sequence analysis allowed us to determine the presence of six of the nine motifs already reported for other ICK/ KRP proteins (Torres Acosta et al., 2011; Supplementary  Fig. S2A) including motifs 1 and 2, located in the carboxyl terminus, required for CDK and cyclin D binding (CDKcyclin interacting/inhibiting domain; Fig. 1B).
Motifs 3 and 4 were not found in maize KRPs, since these appear to be exclusive to class A proteins (dicotyledonous). On the other hand, proteins with motifs 5, 6 and 8 are grouped in class C in maize, whereas these motifs in rice proteins are distributed in classes B and C (Torres Acosta et al., 2011). Motif 7 is present in proteins of classes B and C, except KRP3, which only contains motifs 1 and 2 (Fig. 1B).
This analysis shows that half of the maize protein sequences have a nuclear localization sequence (NLS) monoor bipartite, only two maize ICK/KRPs have putative PEST sequences and all proteins present several CDK phosphorylation sites (see Supplementary Fig. S2B).

Expression of maize ICK/KRP genes during seed germination and in tissues
Expression of maize ICK/KRP genes was monitored from 0 to 24 h of germination and in different plantlet tissues. Specific primers were designed for each ICK/KRP (RT-PCR, Supplementary Table S3); the level of expression was normalized by using expression of the 18S rDNA gene as control. Since KRP1;2a and KRP1;2b genes are identical, primers amplify both transcripts.
During germination all ICK/KRP genes were expressed at 24 h; expression of KRP1;1 and KRP4;2 seemed not to vary along germination but there was a variable expression for all other genes. KRP1;2, KRP1;3, KRP3, and KRP4;1 were not detected in dry embryos. There was no correlation between the pattern of expression and the phylogenetic class to which every ICK/KRP belongs (Fig. 2).

Inhibition of kinase activity in CycD-CDK complexes by KRP1;1 and KRP4;2 proteins
Previously, we reported differential kinase activity in several CycD-CDK complexes during maize germination (Godínez-Palma et al., 2013), and suggested inhibition of kinase activity by KRPs as a possible mechanism to explain the results obtained. In this paper, two representative members of different phylogenetic classes (KRP1;1, class B and KRP4;2, class C), which are expressed at all germination times, were studied following their inhibitory activity on kinase activity in CycD-CDK complexes.
Kinase activity in CycD-CDK complexes was reduced by KRP1;1 compared with controls receiving BSA (5.0 µg) or not receiving the KRP protein. Activity in complexes formed by CycD2;2-CDK, D4;2-CDK and D6;1-CDK (Fig. 3A, B, D) disappeared when these were incubated with the highest amount of recombinant KRP (5.0 μg), whereas activity in complexes with CycD5;3 was reduced with 2.5 μg and was no longer detectable at the highest concentration (Fig. 3C).

Complex formation between CycsD, CDKA and KRPs
In mammals, the Cip/Kip family of kinase inhibitors associates to Cycs and CDKs forming different heterotrimeric complexes that differ during cell cycle progression and constitute a critical mechanism to control a successful cell proliferation (Plavletich, 1999).
The differential inhibition of kinase activity observed for KRP4;2 suggested that this KRP interacts differentially with CycD-CDK complexes. For this reason, interaction between cyclins D, CDKA and KRPs was studied. In vitro proteinprotein interactions were performed by using recombinant proteins bound to distinct protein tags (CycD2;2-MBP, PeX-CycD6;1, GST-CycD6;1, GST-CDKA, His-KRP1;1, and His-KRP4;2). Recombinant proteins were independently induced with IPTG, and the corresponding soluble fractions were mixed and incubated for 18 h. Then different affinity columns allowed us to retain the specific fusion protein and in this way to identify, using specific antibodies, the corresponding coeluting protein(s). Experimental conditions and controls are shown in Supplementary Table S4 Fig. S7B), since interaction assays indicated that these proteins coeluted.
CycD2;2-MBP/His-CDKA or GST-CycD6;1/His-CDKA recombinant complexes were used to in vitro phosphorylate His-KRP4;2 using cold ATP. His-KRP4;2 was then purified using a phospho-affinity resin; Cak1 was not added in this kinase assay to avoid the presence of a phosphorylated CDK. Phosphorylation was demonstrated by western blot using an anti-pT-P/pS-P antibody (see Supplementary Fig.  S8A, B). ;1-CDK complexes. Lane 1: protein molecular mass markers. Lane 2: immunoprecipitate (IP) using the corresponding anti-CycD antibody without recombinant KRP. Lane 3: IP using the corresponding anti-CycD antibody incubated with 5.0 µg BSA. Lanes 4-6: IPs using the corresponding anti-CycD antibodies incubated with 0.25, 2.5 or 5.0 µg recombinant KRP1;1, respectively. Lanes 7-9: IPs using anti-CycD antibodies incubated with 0.25, 2.5 or 5.0 µg recombinant KRP4;2, respectively. Lane 10: IP using anti-CycD antibodies without GST-Ct-RBR or KRP added. Coomassie Blue stained gels were used as loading control. Densitometry analysis was performed relating band intensity of all samples to the intensity of the loading control and then to the positive control band. Each bar represents the mean±SE from three independent biological replicates. *Statistically significant value (P<0.001) compared with control.

Differential KRP4;2 phosphorylation by CycD-CDK complexes modifies its inhibitory capacity
To evaluate the inhibitory capacity of phosphorylated His-KRP4;2 by either of the kinase complexes, p-His-KRP4;2 was added to kinase assays using anti-CycD2;2-CDK or anti-CycD6;1-CDK immunoprecipitates from embryo axes.

The family of maize ICK/KRP proteins: characterization
Multiple ICK/KRPs are present in plants, and maize contains at least nine different genes. These have been divided into two classes according to a previously defined classification, five of them in class B (ICK2/KRP1;1, KRP1;2a, KRP1;2b, KRP1;3, and KRP3, specific to monocotyledonous plants), and four in class C (ICK4/KRP4;1, ICK1/KRP4;2, ICK3/ KRP5;1, and KRP5;2, present in both mono-and dicotyledonous plants) and none in class A (only dicotyledonous; Torres Acosta et al., 2011). The different protein motifs present in every class could be related to the function they perform in different tissues or at different physiological stages. As expected, all maize ICK/KRPs conserve motifs 1 and 2, essential for interaction with CDKs, kinase activity inhibition and binding to CycsD, both localized to the carboxyl end (CID) (Wang et al., 1997;Lui et al., 2000;Zhou et al., 2003a, b). No information exists as to the function of the other motifs.
Half of the maize ICK/KRP proteins possess an NLS sequence (mono-or bipartite) and most of them conserve motif 7 (except KRP3), both characteristics involved in subcellular localization (Zhou et al., 2003a;Jakoby et al., 2006;Zhou et al., 2006), and therefore it is very likely that maize ICK/KRPs are localized to the nucleus, as already reported for all Arabidopsis KRPs, whether they have NLS or not Bird et al., 2007;Torres Acosta et al., 2011).
The presence of putative PEST sites in KRPs suggests degradation via the ubiquitin-proteasome system (Rogers et al., 1986;Schnittger et al., 2003), although this sequence by itself does not guarantee ubiquitination. Only two maize ICK/ KRPs (KRP1;3 and KRP3) contain a PEST sequence; however, there is no evidence for ubiquitin-mediated degradation, or any other mechanism, of maize KRPs. In Arabidopsis, ubiquitin-dependent degradation of ICK4/KRP6 and ICK5/ KRP7 seems to control male gametogenesis (Lui et al., 2000;Kim et al., 2008); however, only ICK4/KRP6 has a PEST site (Torres Acosta et al., 2011); also, a motif in ICK1/KRP1 is critical for its stability but shows an atypical ubiquitination sequence, suggesting a degradation mechanism independent of SCF (Li et al., 2016). On the other hand, all maize ICK/KRP proteins contain CDK phosphorylation sites. In Arabidopsis ICK2/KRP2 can be phosphorylated by both CDKA;1 and CDKB1;1, modification that reduces its stability (Verkest et al., 2005). Results shown in this paper also demonstrate in vitro maize ICK/ KRP phosphorylation by CycD-CDK complexes; however, the role of this phosphorylation may be different from that suggested for Arabidopsis KRPs, as will be discussed below.

Expression of maize ICK/KRP proteins
All maize ICK/KRP genes are expressed in different, proliferating or differentiating tissues, with different patterns, notwithstanding the class they belong to perhaps indicating that they participate both in cell cycle arrest and in cell differentiation, as has already been suggested in other plants (Wang et al., 1997;Jasinski et al., 2002a). In Arabidopsis, expression of ICK1/KRP1 and ICK2/KRP2 increases when the cell cycle is arrested (Menges et al., 2005) and overexpression of ICK1/KRP1 causes endoreduplication events Verkest et al., 2005).
Expression of ICK/KRPs in maize during germination shows that some of them accumulate during the early hours, others are expressed at all times and all are present at 24 h of germination. Evidence indicates that during maize germination, the S phase starts by 12 h of imbibition and the first mitotic figures are observed after 24 h (Baíza et al., 1989;Herrera et al., 2000); thus, KRPs might be involved in both the G1-S and G2-M transitions during this developmental process. In Arabidopsis, KRP3 and KRP5 show a peak of expression in S phase, KRP4 during G2, KRP1 in the G2-M transition, KRP6 during the M-G1 transition, whereas KRP2 and KRP7 remain at constant levels during the cell cycle (Menges et al., 2005).

Inhibition of kinase activity in CycD-CDK complexes by KRPs
Members of the two different classes of maize KRPs, KRP1;1 (class B) and KRP4;2 (class C) inhibited the associated kinase activity in CycD2;2-CDK, D4;2-CDK, D5;3-CDK and D6;1-CDK complexes. KRP1;1 inhibits activity in CycD2;2-CDK, D4;2-CDK and D6;1-CDK complexes at the highest concentration used, whereas inhibition of activity in CycD5;3-CDK was achieved with a lower inhibitor concentration. Since all CycsD studied here bind A or B type CDKs (Godínez-Palma et al., 2013;Vázquez-Ramos, unpublished data) the kinase activity measured must be due to complexes containing either CDK. CycD5;3-CDK seems to be more sensitive to KRP inhibition and, incidentally, CycD5;3 binds mainly to CDKA during early maize germination (Godínez-Palma et al., 2013); this suggests that this higher sensitivity might be due to preferential association to only one and not both types of CDKs.
Similarly, kinase activity inhibition by His-KRP4;2 was only visible at the highest concentration for CDK complexes containing CycsD2;2, D4;2 and D5;3; however, activity in CycD6;1-CDK complexes was not inhibited at any concentration used. CycD6;1 is peculiar because it lacks the LXCXE motif, a characteristic sequence in all CycsD, related to retinoblastoma-related (RBR) protein binding. It has been demonstrated for Arabidopsis and maize CycD6;1 proteins that this motif is not necessary for RBR phosphorylation (Cruz-Ramírez, et al., 2012;Zamora-Zaragoza, 2015, unpublished data). Thus, our results suggest that the difference of kinase activity inhibition by maize ICK/KRPs might be due to a different KRP association capacity to CycD-CDK complexes. CycD2;2-MBP binds GST-CDKA, His-KRP1;1 and His-KRP4;2, while PeX-CycD6;1 binds GST-CDKA and His-KRP1;1 but not His-KRP4;2, and this could determine the degree of inhibitory activity; i.e. inhibition may require KRP binding to both the cyclin and the CDK proteins.
It is puzzling that kinase activity inhibition for most CycD-CDK complexes is achieved when KRP concentration is just doubled (from 2.5 to 5 µg); in fact, addition of 3.75 µg already shows some inhibition (data not shown). We do not know if this is due to a certain stoichiometric relationship or if it is due to some structural modification of KRPs, perhaps a structural change due to a gradual KRP phosphorylation that finally makes it a more potent inhibitor (see below).
Preliminary results suggest that maize CycD6;1 binds Zeama-RBR in different sites (Zamora-Zaragoza, unpublished data); perhaps this difference of CycD6;1 association with substrates, compared with the association characteristics of canonical cyclins D, could explain the difference in interaction between PeX-CycD6;1 and the two KRPs: CycD6;1 may bind other sites than the CID in KRPs, perhaps a sequence present in the amino region of His-KRP1;1, which is absent in the shorter His-KRP4;2 (see Fig. 1). In tomato KRP1, there appears to be a sequence in the central region of the protein that is bound by CycD3 (Nafati et al., 2010).

Phosphorylation of KRPs modify their inhibitory capacity
All maize ICK/KRPs have at least one putative CDK phosphorylation site and results demonstrated that all CycD-CDK complexes used here phosphorylated recombinant His-KRP1;1 and His-KRP4;2 proteins, besides phosphorylating Zeama-GST-Ct-RBR (Fig 5). In Arabidopsis ICK/ KRPs and also in yeast Sic1p protein, phosphorylation by Cyc-CDK complexes promotes protein degradation, an essential mechanism for control and progression of the cell cycle (Nishizawa et al., 1998;Verkest et al., 2005). In this context, that maize KRPs are phosphorylated is not surprising.
What is relevant in maize KRP phosphorylation is that the inhibitory activity can be selectively enhanced. His-KRP4;2 phosphorylation by the different recombinant kinase complexes has a differential effect on kinase activity in immunoprecipitated CycD-CDK complexes from embryo axes. His-KRP4;2 phosphorylation by a recombinant CycD2;2-MBP-His-CDKA complex increases kinase activity inhibition of CycD-CDK complexes, even at lower KRP concentrations. Recombinant alfalfa KRP is phosphorylated by a calmodulin-dependent kinase, increasing KRP inhibitory activity on MtCycA;2-CDKA;1 (Pettkó-Szandtner et al., 2006). There is no information, to our knowledge, that in alfalfa, or any other plant, Cyc-CDK complex phosphorylates and activates KRPs.
On the other hand, His-KRP4;2 phosphorylated by a recombinant GST-CycD6;1-His-CDKA complex behaves differently, having little, or no inhibitory activity against CycD-CDK complexes at all concentrations tested. These results suggest that the different CycD-CDK complexes phosphorylate His-KRP4;2 in different residues, and depending on which is phosphorylated, the inhibitory capacity of His-KRP4;2 could be enhanced or reduced. In this context, it is relevant that phosphorylated KRPs can be re-phosphorylated by another CycD-CDK, as indicated in Fig. 6, giving evidence that more than one residue could be modified.
A differential phosphorylation of His-KRP4;2 could be the result of the direct or indirect interaction of this KRP with CycsD; interaction of CycD2;2-MBP and His-KRP4;2 could result in His-CDKA phosphorylation of canonical residues, whereas the indirect interaction of CycD6;1 and His-KRP4;2 could produce phosphorylation in other residues, which modify the inhibitory capacity of His-KRP4;2.
Work with budding yeast has demonstrated that Sic1p displays a differential binding affinity toward G1 cyclins (CLNs) and S-M cyclins (CLBs) (Kõivomägi et al., 2011). CDK1-CLNs bind but are not inhibited by Sic1p; instead Sic1p is phosphorylated at multiple sites targeting it to destruction (Cross et al., 2007), which in turn causes the release of the CLB5-CDK1 complex from Sic1p inhibitory activity, thus triggering S phase onset (Verma et al., 1997;Nash et al., 2001). In maize, all cyclins D studied are present through the whole cell cycle process that takes place during germination (De Jesús Juárez et al., 2008;Lara-Núñez et al., 2008;Godínez-Palma et al., 2013;Vázquez-Ramos, unpublished data). In this context, it is very interesting that the CycD6;1-CDK complex is not inhibited by KRP4;2 but does phosphorylate it, somehow resembling the situation in budding yeast, perhaps establishing a role for this kinase complex in promoting cell cycle advance. Therefore, it is tempting to speculate that phosphorylation of KRPs in different residues, or by different CycD-CDK complexes, would be responsible for their association, activation, inhibition or destruction. This could be part of the mechanism by which cell cycle transitions are controlled and would depend on the appearance of the different CycD-CDKs.
It will be very important to understand which residues are phosphorylated in every KRP and if phosphorylation of other residues modifies their stability or inhibitory capacity.
Finally, the great variety of KRPs and also of CycD-CDK complexes point to a complex regulatory network that perhaps is related to the developmental needs of different tissues or responds to the different environmental conditions to which plants have to adapt.