A cdc2-like kinase phosphorylates histone H1 in the amitotic macronucleus of Tetrahymena.

Abstract

Genetic and biochemical studies have shown that cdc2 protein kinase plays a pivotal role in a highly conserved mechanism controlling the entry of cells into mitosis. It is generally believed that one function of cdc2 kinase is to phosphorylate histone H1 which in turn promotes mitotic chromosome condensation. However, direct evidence linking H1 phosphorylation to mitotic chromatin condensation is limited and the exact cellular function(s) of H1 phosphorylation remains unclear. In this study, we show that mammalian cdc2 kinase phosphorylates H1 from the amitotic macronucleus of Tetrahymena with remarkable fidelity. Furthermore, we demonstrate that macronuclei from Tetrahymena contain a growth‐associated H1 kinase activity which closely resembles cdc2 kinase from other eukaryotes. Using polyclonal antibodies raised against yeast p34cdc2, we have detected a 36 kd immunoactive polypeptide in macronuclei which binds to Suc1 (p13)‐coated beads and closely follows H1 kinase activity. Since macronuclei divide without mitotic chromosome condensation, these data demonstrate that H1 phosphorylation by cdc2 kinase may be necessary, but is not sufficient to promote mitotic chromatin condensation. The fact that an activity which strongly resembles mammalian cdc2 kinase is active during cell growth in a nucleus which does not undergo mitosis and chromosome condensation suggests that other factors are needed for a true mitotic division to occur. These data also reinforce the notion that H1 phosphorylation has important functions outside mitosis both in Tetrahymena and in mammalian cells.

Four distinct and unusual linker proteins in a mitotically dividing nucleus are derived from a 71-kilodalton polyprotein, lack p34cdc2 sites, and contain protein kinase A sites.

“…In contrast, Roth et al (53) have presented evidence that the amitotically dividing Tetrahymena macronucleus contains an Hi kinase that is immunologically and enzymatically similar to p34cdc2 kinase and that macronuclear Hi is phosphorylated by this kinase at sites similar to those recognized by p34cdc2 kinase in mammalian Hi. Roth et al (53) have suggested that Hi phosphorylation by p34cdc2 kinase may be necessary but not sufficient for mitotic chromosome condensation. However, the complete absence of p34cdc2 kinase sites in Mic LH argues that while p34Cd2 kinase action on Hi might be required for some cell cycle events, it may not be necessary for chromosome condensation per se.…”

“…Because Hi has been postulated to play an important role in higher-order chromatin structure (see reference 59 for a review), and macronuclei do not undergo mitotic chromosome condensation, it was suggested that the central globular core of Hi might be involved in-this process (62). Recently, it has been shown (53) that macronuclear Hi is phosphorylated by a p34cdc2-ike kinase. Since Hi phosphorylation by this kinase is thought to play a role in mitotic chromosome condensation, its occurrence in the amitotic macronucleus is problematic (see references 53 and 52 for discussions).…”

Proteolytic removal of core histone amino termini and dephosphorylation of histone H1 correlate with the formation of condensed chromatin and transcriptional silencing during Tetrahymena macronuclear development.

“…Although this finding seems to contradict the popular idea that H1 phosphorylation leads to chromatin condensation during mitosis (Bradbury et al 1974a, b), direct evidence linking H1 phosphorylation to chromatin condensation is lacking (for discussion, see Roth et al 1988Roth et al , 1991. Furthermore, H1 dephosphorylation has been observed in other biological systems where condensed chromatin and transcriptional silencing occur (Sung 1977;Green and Poccia 1985;).…”

Identification and Mutation of Phosphorylation Sites in a Linker Histone

“…Like H1 of multicellular eukaryotes, macronuclear H1 is a substrate for p34 cdc2 in vitro, and we have presented evidence previously that suggests that this H1 is phosphorylated by a homolog of Cdc2 in vivo (21,22). However, unlike many eukaryotes in which high levels of H1 phosphorylation occur mainly during mitosis, the majority of macronuclear H1 molecules are highly phosphorylated in unsynchronized, growing cultures of Tetrahymena (19,(21)(22)(23). Given the absence of mitotic chromosome condensation in the amitotically dividing macronucleus (18), these differences likely reflect the high percentage of the macronuclear genome that is transcribed (24).…”