Archives

  • 2018-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • 2021-01
  • 2021-02
  • 2021-03
  • 2021-04
  • 2021-05
  • 2021-06
  • 2021-07
  • 2021-08
  • 2021-09
  • 2021-10
  • 2021-11
  • 2021-12
  • 2022-01
  • 2022-02
  • 2022-03
  • 2022-04
  • 2022-05
  • 2022-06
  • 2022-07
  • 2022-08
  • 2022-09
  • 2022-10
  • 2022-11
  • 2022-12
  • 2023-01
  • 2023-02
  • 2023-03
  • 2023-04
  • 2023-05
  • 2023-06
  • 2023-08
  • 2023-09
  • 2023-10
  • 2023-11
  • 2023-12
  • 2024-01
  • 2024-02
  • 2024-03
  • 2024-04
  • 2024-05

    • In maize embryo axes tissues the co elution

    2020-07-27

    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 3ma 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). 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, antibodies 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.