PHOSPHORYLATION OF LINKER HISTONES BY A PROTEIN-KINASE A-LIKE ACTIVITY IN MITOTIC NUCLEI

Micronuclear linker histones of the ciliated protozoan, Tetrahymena th ermophila, are extensively phosphorylated in vivo. Each of these polyp eptides, alpha, beta, gamma, and delta, contains sites for phosphoryla tion by cyclic-AMP dependent protein kinase (PKA) but not Cdc2 kinase, and some data have been presented implicating PKA kinase in their pho sphorylation in vitro and in vivo (Sweet, M. T., and Allis, C. D. (199 3) Chromosoma 102, 637-647; Sweet, M. T., Jones, K., and Allis, C. D. (1996) J. Cell Biol., in press). In this report we have extended these analyses by showing that Cdc2 and PKA kinase are not evenly distribut ed between micro- and macronuclei. Macronuclei, but not micronuclei, c ontain a 36-kDa polypeptide that is immunoreactive with p34(Cdc2) anti bodies. In contrast, a 40-kDa polypeptide is detected with PKA antibod ies in micronuclei, that is not detected in macronuclei. In support, e xtracts from micronuclei, but not macronuclei, contain a kinase activi ty that resembles some, but not all, characteristics of PKA from other sources. Immunodepletion experiments using anti-PKA antibodies show t hat a 40-kDa polypeptide can be specifically removed from these extrac ts with a concomitant loss in kinase activity. Microsequence analyses of delta demonstrate that this linker histone is phosphorylated in viv o on two PKA consensus sequences located in its carboxyl-terminal doma in, an optimum PKA consensus sequence, Arg-Lys-Asn-Ser, and a less opt imal PKA sequence, Lys-Ser-Ser-Val. Collectively, these results sugges t that PKA or a PKA-like kinase is responsible for the phosphorylation of linker histone in mitotically dividing micronuclei. In contrast, m acronuclei, which divide amitotically, phosphorylate linker histone H1 using a distinct, Cdc2-like kinase.