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  • During DNA replication p protein binds PCNA in

    2019-12-02

    During DNA replication p21 protein binds PCNA, in its role as processivity factor, to stop replication when there is DNA damage, and apparently p21 also binds PCNA when this is in complex with Cyc/CDKs [11]. In plants, KRPs (functional analogs of p21), inhibit kinase activity in CycD/CDKs complexes [[19], [20], [21], [22],28]. Here we show that two members of the maize KRP family (KRPs 1;1 and 4;2) directly bind to PCNA both in vitro and in vivo during maize germination. When the PCNA-associated kinase activity from maize embryo axes is exposed to either KRP, activity is considerably reduced indicating that the kinase associated to PCNA is of the CDK type and that a quaternary complex can be formed, even if it is a transient one. Whether the KRPs bind to the Cyc/CDK complex, to PCNA or to both, is still not known. Direct binding of PCNA to KRPs may also take place when PCNA is involved in DNA replication, similar to p21 and PCNA in animal cells, but we have no evidence for this. PCNA-associated kinase activity is also inhibited by the CDK-specific inhibitor Roscovitine. During maize germination, Ramipril against KRPs co-precipitated PCNA. Following kinase activity in PCNA immunoprecipitates from maize embryo axes imbibed for different periods, a differential inhibitory activity was found. Immunoprecipitates from 0 h-imbibed axes showed that KRPs decrease activity by only 20% whereas the inhibitory activity was 50% at 12 h and 70% at 24 h of germination. This differential inhibitory activity may be due to variations in the composition of the Cyc/CDK complexes, i.e., perhaps some complexes contain d-Cycs, or CDKs that are less susceptible to KRPs and they appear at different times during germination. Previous results have shown that maize CycD6;1/CDKs complexes are refractory to inhibition by KRP4;2 [21]. The inhibitory capacity seems to depend on the phosphorylation status of KRPs [43] and it could be possible that this phosphorylating enzyme is not present at 0 h, but, appears at later germination times. The presence of other accompanying proteins in 0 h kinase complexes that precluded KRP inhibition is also likely. The presence of PCNA co-eluting with Cycs/CDKs at different molecular mass fractions during germination suggests changes in the accompanying proteins that may affect kinase activity or KRP inhibitory capacity. The S phase during maize germination starts by 12 h after imbibition and by 24 h cells are entering to the M phase [23,26], so it would be expected that the molecular status of PCNA gradually changed from being part of G1 kinase complexes to be the processivity factor for DNA polymerases. A role for PCNA in G2/M is not known and thus its binding capacity to a putative G2/M protein as CDKB1;1 is worth exploring.
    Conflict of interest
    Author contributions
    Acknowledgements This work was supported by Consejo Nacional de Ciencia y Tecnología (grant number CB 220661); Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica (grant numbers IN215316 and IA204817); Programa de Apoyo a Investigación y Posgrado (grant numbers 5000-9124 and 5000-9130); Consejo Nacional de Ciencia y Tecnología–PhD grant (to S.M.G.A. and J.A.M.) and Research Assistant FQ-UNAM grant (to A. T. L. E.).
    Introduction Cyclin-dependent kinases (CDKs) are serine/threonine kinases that belong to the CMGC group of kinome and their activities are regulated by cyclin binding and inhibitors from the CIP/KIP and INK4 families in response to several extra- and intracellular signals [1], [2]. Deregulation of CDKs is frequently associated with human cancers [3], [4], and therefore, the development of synthetic inhibitors for therapeutic purposes has recently resulted in the approval of palbociclib (PD0332991, Ibrance®) and ribociclib (LEE011, Kisqali®) for the treatment of advanced breast cancer [5], [6], [7]. The CDK family comprises 20 members that are responsible for the phosphorylation of substrates that are important for cell cycle progression and transcription. Several studies have revealed that CDKs can also mediate steroid receptor phosphorylation at multiple sites, including the androgen receptor [8], [9], [10].