Protein kinase CK2 differentially phosphorylates maize chromosomal high mobility group B (HMGB) proteins modulating their stability and DNA interactions.

The high mobility group (HMG) proteins of the HMGB family are architectural factors in eukaryotic chromatin, which are involved in the regulation of various DNA-dependent processes. We have examined the post-translational modifications of five HMGB proteins from maize suspension cultured cells, revealing that HMGB1 and HMGB2/3, but not HMGB4 and HMGB5, are phosphorylated by protein kinase CK2. The phosphorylation sites have been mapped to the acidic C-terminal domains by analysis of tryptic peptides derived from HMGB1 and HMGB2/3 using nanospray ion trap mass spectrometry. In native HMGB1, Ser(149) is constitutively phosphorylated, whereas Ser(133) and Ser(136) are differentially phosphorylated. The functional significance of the CK2-mediated phosphorylation of HMGB proteins was analyzed by circular dichroism measurements showing that the phosphorylation increases the thermal stability of the HMGB proteins. Electrophoretic mobility shift assays demonstrate that the phosphorylation reduces the affinity of the HMGB proteins for linear DNA. The specific recognition of DNA minicircles is not affected by the phosphorylation, but a different pattern of protein-DNA complexes is formed. Collectively, these findings show that phosphorylation of residues within the acidic C-terminal domain of the HMGB proteins can modulate protein stability and the DNA binding properties of the HMGB proteins.     

Binding of high-mobility-group proteins HMG 14 and HMG 17 to DNA and histone H1 as influenced by phosphorylation

We have used affinity chromatography to study the effects of phosphorylation of calf thymus high-mobility-group proteins HMG 14 and HMG 17 on their binding properties towards calf thymus single- and double-stranded DNA and histone H1. Without in vitro phosphorylation, HMG 14 and HMG17 eluted from doble-stranded DNA-columns at 200 mM NaCl. HMG 14 was released from single-stranded DNA-column at 300 mM NaCl and from H1-column at 130 mM NaCl, whereas the corresponding values for HMG 17 were 230 mM and 20 mM, respectively. Phosphorylation of HMG 14 and HMG 17 by cAMP-dependent protein kinase (A-kinase) decreased markedly their affinity (270 mM and 200 mM NaCl, respectively) for single-stranded DNA, whereas HMG 14 phosphorylated by nuclear protein kinase II (NII-kinase) eluted only slightly (290 mM NaCl) ahead of the unphosphorylated protein. HMG 14 phosphorylated by both A-kinase and NII-kinase eluted from double-stranded DNA-columns almost identically (190 mM NaCl) with the unphosphorylated protein. Interestingly, phosphorylation of HMG 14 by NII-kinase increased considerably its affinity for histone H1 and the phosphorylated protein eluted at 200 mM NaCl. Phosphorylation of HMG 14 by A-kinase did not alter its interaction towards histone H1. These results indicate that modification of HMG 14 by phosphorylation at specific sites may have profound effects on its binding properties towards DNA and histone H1, and that HMG 17 has much weaker affinity for single-stranded DNA and histone H1 than HMG 14.     

Multiple Interactions of the Intrinsically Disordered Region between the Helicase and Nuclease Domains of the Archaeal Hef Protein

Hef is an archaeal protein that probably functions mainly in stalled replication fork repair. The presence of an unstructured region was predicted between the two distinct domains of the Hef protein. We analyzed the interdomain region of Thermococcus kodakarensis Hef and demonstrated its disordered structure by CD, NMR, and high speed atomic force microscopy (AFM). To investigate the functions of this intrinsically disordered region (IDR), we screened for proteins interacting with the IDR of Hef by a yeast two-hybrid method, and 10 candidate proteins were obtained. We found that PCNA1 and a RecJ-like protein specifically bind to the IDR in vitro. These results suggested that the Hef protein interacts with several different proteins that work together in the pathways downstream from stalled replication fork repair by converting the IDR structure depending on the partner protein.     

Phosphorylation of nuclear proteins

Many nuclear proteins are phosphorylated: they range from enzymes to several structural proteins such as histones, non-histone chromosomal proteins and the nuclear lamins. The pattern of phosphorylation varies through the cell cycle. Although histone H1 is phosphorylated during interphase its phosphorylation increases sharply during mitosis. Histone H3, chromosomal protein HMG 14 and lamins A, B and C all show reversible phosphorylation during mitosis. Several nuclear kinases have been characterized, including one that increases during mitosis and phosphorylates H1 in vitro. Factors have been demonstrated in maturing amphibian oocytes and mitotic mammalian cells that induce chromosome condensation and breakdown of the nuclear membrane. The possibility that they are autocatalytic protein kinases is considered. The location of histone phosphorylation sites within the nucleosome is consistent with a role for phosphorylation in modulating chromatin folding.     

Phosphorylation of high-mobility-group protein 14 by two specific kinases modifies its interaction with histone oligomers in free solution

Chromosomal protein HMG14 can be specifically phosphorylated by the cyclic AMP-dependent protein kinase at the N-terminus and by casein kinase 2 at the acidic C-terminus. Under the same conditions used for HMG14, HMG17 is not significantly phosphorylated by either of the two kinases. Further, we have studied the effect of phosphorylation by these kinases on the interaction of HMG14 with histone oligomers, using chemical cross-linking. Our results indicate that the phosphorylation of HMG14 by casein kinase 2 enhances its interaction with histone oligomers in free solution, whereas a minor effect was observed by phosphorylation with cyclic AMP-dependent protein kinase. In contrast, HMG17 does not interact at all with any histone oligomer in free solution under the conditions used. To gain insight into the possible effect that phosphorylation may play in vivo, the pattern of distribution among different chromatin fractions was analysed. It was found that, although phosphorylation of HMG14 by both kinases allowed reconstitution of HMG14 to chromatin, the patterns obtained showed some slight differences.     

A one-step preparative method for separating SER 6-phosphorylated HMG 14 from unphosphorylated HMG 14 and in vitro phosphorylation reaction components

While clear evidence exists for the regulation of the phosphorylation of the very basic high mobility group (HMG) and histone chromatin proteins, the physiological role of their phosphorylation remains poorly understood. Elucidation of these roles has been difficult, in part, because of the inability to obtain sufficient quantities of purified phosphorylated derivatives. We have used Mono S cation-exchange chromatography to prepare milligram quantities of pure Ser 6-phosphorylated HMG 14 (Ser 6-PO4-HMG) from unphosphorylated Mono S-purified calf thymus HMG 14 following in vitro phosphorylation with cAMP-dependent protein kinase (A-kinase). In one step, this technique separates the phosphorylated derivative from A-kinase, ATP, unphosphorylated HMG 14, and a minor phosphorylated by-product which evidence suggests may be the previously reported Ser 6, 24-diphospho-HMG 14. Mono S chromatography also enhances the purity of calf thymus HMG 14 prepared by perchloric acid extraction, acetone and ethanol precipitations, and CM-Sephadex chromatography. In addition, it permits the detection of apparent microheterogenous forms of both unphosphorylated and Ser 6-PO4-HMG 14. The significant reductions in binding affinity resulting from the incorporation of phosphate groups into HMG 14 suggest that Mono S chromatography could have more general application in the isolation of phosphorylated derivatives of other basic proteins, including other chromatin-associated DNA-binding proteins which are known to undergo specific phosphorylation. It would especially be useful when the proteins and their phosphorylated derivatives bind more tightly to Mono S than the kinases used for their phosphorylation.     

Consecutive steps of phosphorylation affect conformation and DNA binding of the chironomus high mobility group A protein

The high mobility group (HMG) proteins of the AT-hook family (HMGA) lie downstream in regulatory networks with protein kinase C, Cdc2 kinase, MAP kinase, and casein kinase 2 (CK2) as final effectors. In the cells of the midge Chironomus, almost all of the HMGA protein (cHMGA) is phosphorylated by CK2 at two adjacent sites. 40% of the protein population is additionally modified by MAP kinase. Using spectroscopic and protein footprinting techniques, we analyzed how individual and consecutive steps of phosphorylation change the conformation of an HMGA protein and affect its contacts with poly(dA-dT).poly(dA-dT) and a fragment of the interferon-beta promoter. We demonstrate that phosphorylation of cHMGA by CK2 alters its conformation and modulates its DNA binding properties such that a subsequent phosphorylation by Cdc2 kinase changes the organization of the protein-DNA complex. In contrast, consecutive phosphorylation by MAP kinase, which results in a dramatic change in cHMGA conformation, has no direct effect on the complex. Because the phosphorylation of the HMGA proteins attenuates binding affinity and reduces the extent of contacts between the DNA and protein, it is likely that this process mirrors the dynamics and diversity of regulatory processes in chromatin.     

Histone phosphorylation during sea urchin development

Studies on histone phosphorylation during transitions in chromatin structure occurringin vivoduring spermatogenesis and early embryogenesis in sea urchins are reviewed and evaluated in the light of recent studies on histone phosphorylation occurring during chromatin synthesis in frog egg extractsin vitroand evidence that protein kinases and phosphatases play direct roles in the regulation of cellular structure. Sperm-specific histone variants Sp H1 and Sp H2B are maintained as phosphorylated derivatives N and O/P throughout spermatogenesis and early embryogenesis and egg specific histone variants CS H1 and CS H2A are phosphorylated during early embryogenesis. These developmental correlations provide clues about the roles of histone phosphorylation in control of chromatin structurein vivoand provide a basis for the interpretation of data obtained from in-vitro sperm chromatin remodeling in egg extracts and from biochemical studies on the effects of histone phosphorylation on DNA binding. The potential consequences for chromatin structure of the various histone phosphorylation events observed in sea urchins and frog egg extracts are discussed.