A PURIFIED 124‐kDa OAT PHYTOCHROME DOES NOT POSSESS A PROTEIN KINASE ACTIVITY*

Abstract

The presence of protein kinase activity in the purified phytochrome preparations [Wong, et al. (1986) J. Biol. Chem. 261, 12089-12097] has been re-examined. The phytochrome preparations having SAR (specific absorbance ratio, A668/A280 for the Pr form as a measure of phytochrome purity) values of greater than 0.95 were homogeneous on SDS gel, but could be further purified to a SAR value of 1.07 by repeated gel filtrations on a Bio-Gel A-0.5 m column. The protein kinase activity remained in the phytochrome preparations having SAR values less than 1.05, but it became undetectable in the phytochrome preparation with a SAR value of 1.07. Two dimensional gel electrophoresis of the phytochrome preparation (SAR, 0.89) showed that a phytochrome band with pl 5.8 had no kinase activity. Phosphorylating activity of the protein kinase was enhanced to some extent by polycations, polylysine and histone. Phytochrome served as a good substrate for this enzyme. The present data indicate that phytochrome has no intrinsic protein kinase activity, but a protein kinase is present in highly purified phytochrome preparations.

Pr-Specific Phytochrome Phosphorylation in Vitro by a Protein Kinase Present in Anti-Phytochrome Maize Immunoprecipitates

“…For example, a co-immunoprecipitated protein could modify the phytochrome-phosphorylating activity, antibody binding might affect phytochrome conformation, or rapid extraction of immunoprecipitates might prevent a modification of protein kinase activity that occurred during phytochrome purification. Polycation stimulation of activity is characteristic of PAPK , but may be negligible in some preparations (Kim et al, 1989) and was not observed here. However, the highly basic IgG proteins present in all protein phosphorylation assays may have acted as a polycationic stimulus.…”

“…Feldman, unpublished results), indicating that the immunoprecipitated protein kinase is charge sensitive like that copurified with phytochrome. Phytochrome immunoprecipitates did not possess appreciable histone kinase activity, although this was reported in purified phytochrome preparations (Kim et al, 1989;. It has been suggested that lack of specificity of kinase activity toward phytochrome in purified preparations is due to loss of kinase regulatory function during extraction (Kim et al, 1989).…”

“…Vol. 105, 1994 activity is not intrinsic to the phytochrome molecule (Grimm et al, 1989;Kim et al, 1989). It is difficult in such studies to completely eliminate the possibility of loss of intrinsic protein kinase activity by denaturation during separation procedures.…”

“…First, protein kinase activity has been found to be associated with purified phytochrome by severa1 research groups (Grimm et al, 1989;Kim et al, 1989;, although it is unclear if these groups were examining the same kinase activity. Since kinase activity could not be found apart from phytochrome, the activity appears to be either intrinsic to the phytochrome polypeptide or residing in another polypeptide that is tightly bound to phytochrome and that requires phytochrome for activity (Kim et al, 1989;. However, in other studies phytochrome without protein kinase activity could be separated from phytochrome with protein kinase activity, suggesting that the Plant Physiol.…”

Higher-plant phytochrome: “I used to date histidine, but now I prefer serine”

“…11 and references therein), the obvious question was raised: How does one distinguish the possibility that phytochrome itself may be a kinase, from a contaminant that copurifies with phytochrome? Indeed, two groups independently reported that protein kinase activity could be removed from phytochrome by extensive purification (12,13). The Lagarias laboratory countered by noting that the properties of this separated kinase were not the same as they had characterized for their phytochrome preparations (14).…”