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  • Proteins that associate to PCNA are


    Proteins that associate to PCNA are characterized by having conserved motifs as the PCNA-interacting protein (PIP) motif [29,30], its variant PIP-like motif [31,32] or the APIM motif (Alk B homologue 2 PCNA interacting motif [33,34]); for instance, p21 contains a PIP motif [30], and DNA polymerases ɳ and ι have a PIP-like motif [31]. In this work we analyzed the interaction of PCNA with different maize (-)-Huperzine A proteins like CycDs, CDKs and KRPs that contain PIP motifs or variants, show that there is kinase activity in PCNA-Cyc/CDK complexes and that this kinase activity is inhibited by KRPs. Even more, PCNA and KRPs associate both in vitro and in vivo suggesting a role for KRP similar to that of p21Kip1. The inhibitory activity of KRPs towards the PCNA-associated kinase activity increases as germination advances, suggesting an important regulatory role for KRPs.
    Materials and methods
    Discussion PCNA is a homotrimer that provides processivity to replicative DNA polymerases during DNA replication. However, PCNA also appears to function as a platform for G1 proteins since it interacts with G1 Cycs and p21Kip1 proteins in animal cells [9,11]. In plants (Arabidopsis and maize) PCNA also associates to CycDs and CDKs [14,27,28]. In animal cells, PCNA has the capacity to form dimers of trimers [7], whose function is still not well understood. Perhaps the dimer of trimers provides a higher contact surface to bind more than one type of protein at specific metabolic times. Here we show that the quaternary structure of endogenous maize PCNA can be found predominantly as trimers and dimer of trimers during the germination process; recombinant maize His-PCNA protein can also form trimers but apparently the dimer formation is very weak, suggesting that in vivo other cellular elements could facilitate the assembly of the dimer. Interestingly, both the trimer and the dimer of trimers were found in nuclei and in the cytoplasm of maize cells, raising the question of their possible role in cytoplasm. It has been shown that PCNA is capable of binding to various cytoplasmic proteins, i.e., proteins involved in glycolysis, stimulating their activity [40]. Maize PCNA may also perform a similar function; otherwise, it would be convenient to know the structure that PCNA acquires when it is transported to the nucleus. It has been reported recently that in animal cells, CycD3/CDK6 complexes phosphorylate and regulate enzymes of the glycolysis pathway [41] and this could also apply to plant cells; the idea that PCNA may be associated to Cyc/CDKs in cytoplasm, perhaps as a mechanism to contact targets becomes very attractive and worth following. In maize embryo axes tissues, the co-elution of PCNA together with CycD3;1 and CDKs in high molecular mass fractions that vary in size along the germination process was indicative of a possible and probably dynamic association to proteins, being the variation in molecular mass due to the association to different targets at every physiological stage. Cyc/CDK targets could be the RBR protein, E2F Transcription Factors or DNA polymerases, among others [15,42]. Two different methodologies used here, binding to a p13Suc1 resin or PCNA immunoprecipitation have demonstrated that all, CycD3;1, CDKA, CDKB1;1, KRPs and PCNA proteins co-purify, perhaps representing diverse complexes: CycD3;1/CDKs, CycD3;1/CDKs-PCNA, CycD3;1/CDKs-KRPs, CycD3;1/CDKs-PCNA-KRPs, or other variants. CycD3;1/CDKA(B1;1) complexes were previously reported [36] as well as PCNA association to different CycDs and CDKs [[26], [27], [28]]. Since recombinant PCNA can bind directly to CDKA, CDKB1;1, KRP1;1 and KRP4;2, this implies that the presence of ternary or quaternary complexes is likely in vivo. It is noteworthy that Arabidopsis CycD3;1 cannot bind AtPCNA [14], establishing differences between the maize and Arabidopsis proteins. The association to a putative G2-M protein, CDKB1;1 has not been reported before. Proteins that bind to PCNA contain conserved motifs like the PIP sequence. The analysis of the primary structure of maize cell cycle proteins (Table 1) showed that CDKA, CDKB1;1 and KRP4;2 have a canonical PIP box and CycsD, CDKs and KRPs have PIP-like boxes. Thus, the interaction of these proteins with PCNA may be through these sequences. As indicated above, some proteins have two types of motifs, a canonical PIP and a PIP-like (CDKs and KRP4;2a). The recently described APIM motif [33,34] was observed in CycD5;3 a and b and in KRP1;3 (Table 1).