Supplementary Results

Supplementary Results

Effect of polyamines

Highly basic polyamines like spermine and spermidine have been shown to affect enzyme activity differently, depending on the nature of the enzyme and also suggested to influence protein phosphorylation (Levasseur et al. 2004). Different concentrations of spermine and spermidine (0.1- 10 mM) were used to study their effects on banana kinase activity. The kinase activity was declined rapidly in the presence 1 mM spermine and declined further with increasing concentrations of spermine. In contrast to spermine, spermidine inhibited the kinase activity at relatively higher concentrations (4 mM and onwards) (Supplementary Figure S4c).

Characterization of partially purified banana kinase

we carried out in-vitro phosphorylation reaction using partially purified protein kinase fraction and Histone III-S was used as substrate in presence of rATP instead of radiolabeled ATP. Similar reaction was carried out without rATP as control (Supplementary Fig. S5a). After incubation with rATP, the reaction mixtures were separated on 10% SDS-PAGE followed by immunoblotting using anti-phosphotyrosine, anti-phosphoserine and anti-phosphothreonine antibodies respectively. As shown in Supplementary Fig. S5b, anti-phosphoserine and anti-phosphothreonine antibodies specifically recognized the band of phosphorylated Histone III-S, while anti-phosphotyrosine antibody did not show any cross reactivity with the substrate. On the other hand, similar experiment with autophosphorylated protein kinase has revealed that the protein kinase was autophosphorylated almost exclusively on tyrosine residue (Supplementary Fig. S5c). Taken together, these results have indicated that the 41-kDa protein kinase belongs to serine/threonine family of protein kinase and is autophosphorylated on tyrosine residue.

Immunoprecipitation and immunoblotting reveals phosphorylation at the C-terminal region of MA-ACS1

To determine which amino acid residue (s) of MA-ACS1 were phosphorylated, 32P-labeled protein extract from ripe banana fruit was immunoprecipitated with MA-ACS1 antibody and the immuno complex was resolved on 10% SDS-PAGE. The labeled proteins were transferred onto a PVDF membrane and immunoprobed with anti-phosphotyrosine, anti-phosphoserine and anti-phosphothreonine antibodies respectively. As shown in Supplementary Fig. S8a, the anti-phosphoserine antibody specifically recognized the 55-kDa band of MA-ACS1, while anti-phosphotyrosine and anti-phosphothreonine antibodies did not cross-react with the phosphorylated MA-ACS1 present in the labeled fruit protein extract. The 55-kDa band of in vivo phospholabeled MA-ACS1 in the ripe banana fruit pulp protein extract, immunoprecipitated with MA-ACS1 antibody, was specifically recognized by anti-phosphoserine antibody (Supplementary Fig S8b, lane 1). Furthermore, rATP-labeled (cold ATP) recombinant full length MA-ACS1 or N-terminal deletion mutant of MA-ACS1 (lacking 328 amino acid residues from the N-terminal region) were specifically cross reacted with anti-phosphoserine immune serum. Conversely, no cross reactivity was detected with labeled (cold ATP) recombinant mutant MA-ACS1 lacking the C-terminal 158 amino acid residues using anti-phosphoserine immune serum (Supplementary Fig. S8b, lanes 2-4). Anti-phosphotyrosine or anti-phosphothreonine antibodies did not show any cross reactivity with rATP-labeled recombinant MA-ACS1 (data not shown). Taken together, these results indicate that MA-ACS1 was phosphorylated on serine residue (s) at the C-terminal region of the protein.

Supplementary Discussion

Polyamines have been reported to bind some key cellular enzymes to modulate their activity and may influence protein phosphorylation. Casein kinase 2-type protein kinases purified from plants and animal tissues have shown to be stimulated by polyamines at millimolar levels, a concentration range proposed to occur in plant cells (Roux 1993). On the other hand, protein kinase activities isolated from wheat anther tissues have been reported to be inhibited by polyamines (Bothma and Dubery 1991). In isolated pea nuclei, polyamines, particularly spermine has been shown to stimulate the phosphorylation of certain nuclear proteins (Datta et al. 1986). Differential effects of polyamines have been demonstrated on rat thyroid protein kinase activities (Levasseur et al. 2004). In our study, the 41-kDa banana protein kinase showed differential sensitivity to the polyamines spermine and spermidine in terms of phosphorylation of Histone IIIS. The kinase activity was more sensitive to spermine than spermidine. The activity was ~2.5 fold reduced in the presence of 1 mM spermine than that of similar concentration of spermidine. Together, these observations may suggest that polyamine may affect the banana ripe fruit tissue associated protein kinase activity and thus phosphorylation of MA-ACS1 during ripening.

References

Bothma C, Dubery IA (1991) The effects of polyamines on protein kinase activities of wheat (Triticum aestivum) L. anthers. Plant growth regul 10:363-375

Datta N, Hardison LK Roux SJ (1986) Polyamine stimulation of protein phosphorylation in isolated pea nuclei. Plant Physiol 82:681-684

Levasseur S, Henricks L, Poleck T, Friedman Y, Burke G (2004) Differential effects of polyamines on rat thyroid protein kinase activities. J Cell Biochem 28:299-306

Roux SJ (1993) Casein kinase-2-type protein kinases in plants: possible targets of polyamine action during growth regulation. Plant growth regul 12:189-193