Phosphorylation
of non-histone chromosomal proteins
One
of most unsual properties of non-histone proteins in the extensive degree to
which they are phosphorylated. The protein-bound phosphate content of this
fraction as a whole is enough to account for several phosphorylated amino acids
for every hundred residues present, giving the non-histone protein fraction the
highest concentration of phosphorylated polypeptides of any protein fraction in
the cell. All the properties discussed thus far as characterizing the
non-histone protein fraction as a whole (i.e. heterogeneity, tissue/species
specificity, association with active chromatin and changes in association with
alteration in gene activity) apply to the phosphorylated non-histone proteins
as well44, 45, 63. In regard to these properties, especially
striking is the vast number of different experimental system in which changes
in non-histone protein phosphorylation have been observed in association with
changing nuclear metabolism, chromatin structure, and gene activity (Table 3.1).
Such observation have stimulated
interest in isolating and identifying individual phosphorylated non-histone
proteins so that their spesific roles in nuclear functions can be investigated.
In the process, it has become gradually apparent that many different kinds of
non-histone proteins are phosphorylated, and that the physiological roles of
these various proteins may be quite diverse. Thus, among the non-histone
nuclear protein now known to be phosphorylated one can distinguish at least a
dozen different catagories: chromatin associated proteins which influence
transcription, nucleolar proteins, the S-100 (brain-specific) protein, RNA
polymerase and associated factors, DNA-binding protein, poy (A) polymerase,
histone deacetylase, glucocorticoid receptors, nucleosomal proteins (including
HMG proteins), protein associated with heterodisperse nuclear RNA, nuclear
envelope proteins, and nuclear matrix protein. A detailed discussion of the
properties of each of these groups of the phosphorylated proteins is beyond the
scope of this chapter, especially since the subject has been recently reviewed
in depth elsewhere63. In the present context, only a few general
issues raised by this diverse group of phosphorylated proteins will be touched
upon.
The most critical issue regrads the significance
of the fact that these proteins are all phosphorylated. The functional
significance of protein phosphorylation has been most clearly estabilished for
cytoplasmic enzymes such as glycogen phosphorylas, phosphorylase kinase, and
glycogen synthetase, where such modifications serve to either increase or
decrease catalytic activity depending on the enzyme in question27, 49, 51,
78, 88. By analogy it has generally been assumed that phosphorylation
serve to regulate the activities of other proteins known to be phosphorylated,
though in most instances such a regulatory role is yet to be clearly
demonstrated. In the case of the phosphorylated non-histone proteins listed
above,
TABLE 3.1 Summary of
experimental systems in which changes in non-histone protein phosphorylation
have been observed (for detailed reference, see reference 63)
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Growth and
Development
Lymphocytes activity
Avian erytrocytes
Physarium
polycephalum
HeLa cells
Reuben hepatoma cells
Yoshida sarcoma
Sea urchin embryos
Trout embryos
Muscle differentiation
Neuronal and glial cells
Kidney regeneration
Barley germination
Chinese hamster cells (K12)
WI-38 fibroblast
Chick embryo fibroblasts
Hamster kidney cells (BHK21C13)
Testis and epididymis
Uterus
ChO cells (heat shock)
Liver (aging)
Liver (regeneration)
Liver (cell culture)
Landschutz tumor cells (amino acid starvation)
Chemical Agents
Isoproterenol (salivary gland)
Isoproterenol (pineal gland)
Acetylcoline (lymphocytes)
Norephinephrine (glioma)
Carbachol (lymphocytes)
Phenobarbitone (liver)
α-1, 2, 3, 4, 5, 6-Hexachlorocyclohexane (liver)
Morphine (brain)
Hemin (liver)
Vitamin D (kidney, liver)
Prostaglandin (lymphocytes)
2, 4-Dichlorophenoxyacetic acid (soybeans)
Helanalin (Ehrlich ascites)
Cytarabine (salivary gland)
Bleomycin (salivary gland)
Chlorambucil (Yoshida sarkoma)
Melphalan (Yoshida sarkoma)
Cyclophosphamide (Yoshida sarkoma)
Eupahyssopin (Ehrlich ascites)
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Hormonal Stimulation
Testosterone (Prostate)
Antiandrogens (prostate)
Glucocorticoids (Insect salivary glands)
Glucocorticoids (liver)
Glucocorticoids (lymphosarcoma)
Aldosterone (kidney)
Oestradiol (uterus)
Oestradiol (mammary carcinoma)
Oestradiol (oviduct)
Oestradiol (brain)
Prolactin (mammary gland)
Chrionic gonadotropin (ovary)
Calcitonin (bone cells)
Triiodothyronine (liver)
Triiodothyronine (heart)
Thyrotropin (thyroid)
Abscisic acid (Lemna)
Cyclic AMP (rat liver)
Cyclic AMP (rat heart)
Cyclic AMP (lymphocytes)
Cyclic AMP (mammary carcinoma)
Cyclic AMP (adrenal medulla)
Cyclic AMP (salivary gland)
Cyclic AMP (Neurospora)
Cyclic AMP (pineal gland)
Cyclic AMP (lymphocytes)
Cyclic AMP (salivary gland)
Maglinant
and Transformed Cells
Mammary carcinoma
Azo-dye carcinogenesis
Dimethylbenzanthracene carcinogenesis
Methylcholanthrene carcinogenesis
SV-40 transformed fibroblast
Morris hepatoma
Novikoff hepatoma
Friend erythroleukimia
Ehrlich ascites
Walker tumor
Adenovirus transformed cells
Murine sarcoma virus transformed cells
Human leukimia
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Phosphorylation
has been shown to affect activation of gene transcription14, 15,
47, 50 non-histone protein binding to DNA35, 50, 61,
67, poly (A) polymerase activity75, 76, RNA polymerase
activity48 and glucocorticoid binding activity65. For
other phosphorylated non-histone proteins, such as S-100 histone deacetylase,
nucleosomal phosphoprotein, hnRNA-associated phosphoprotein, and nuclear
envelope and nuclear matrix phosphoproteins, no direct evidence concerning the
functional significance of phosphorylation is avaliable. Though there is no
shortage of attractive ideas (e.g. phosphorylation of hnRNA-associated proteins
may be involved in regulating RNA processing and/or transport, phosphorylation
of histone deacetylase may regulate its catalytic activity, phosphorylation of
nucleosomal proteins may influence chromatin structure, etc), one should
exercise extreme caution in such instance where there are no definitive data
avaliable.
The fact that the phosphorylation
state of non-histone proteins influences their ability to stimulate RNA
synthesis and bind to DNA in vitro raises
the question of the possible role of non-histone phosphorylation in the control
of spesific gene expression. This question is difficult to answer unequivocally
because of the severe limitations of the cell-free systems in which such investigations
have been carried out. However, after discussing the remaining properties of
non-histone protein protein fraction, we will return to this general issue in
more detail, discussing both the limitation of the current data implicating
non-histone proteins in specific gene regulation and the prospects for
obtaining more definitive information in the future.
DNA binding of
non-histone chromosomal proteins
Selective
gene expression implies the restriction of some genetic loci and the specific
activation of others. If the non-histone proteins play a role in these
processes, it is reasonable to postulate that the mechanism through which
transcriptional control is achieved will depend, in part, on associations
between these proteins and DNA. In prokaryotes these are several examples of
DNA binding proteins which regulate RNA synthesis, for example lac represormediated inhibitor of
transcription of the lac operon2,
26 and the cAMP receptor-mediated facilitation of initation of lac and
gal transcription4, 104.
Spesific DNA binding non-histone
proteins have been isolated by several groups of workers. In the section we
will discuss some of the properties of these DNA binding proteins which may
have a bearning on their postulated role in the control of gene expression.
Sequence-specific DNA
binding non-histone chromosomal proteins
DNA
binding non-histone proteins have been most commonly isolated by affinity
chromatography on coloums containing immobilized homologous or heterologous
DNAs. Only a small proportion of the proteins in non-histone preparations bind
specifically to homologous DNA46, 92 and many of these are
phosphoproteins43. Considerable confusion exists as to heterogeneity
of these proteins, some groups observing a highly complex population of
non-histone protein43, 89, whereas others do not68, 92,
94. Similarly, most groups have found that non-histones bind selectively
to homologous DNA, though others have found little species-specific binding and
argue that their interaction with DNA has severaly restricted the evolutionary
diversity of these proteins. Such data must generally be considered with some
coution because binding conditions very from group to group, different
non-histone protein preparations are used, in most cases considerable overlap
of proteins in DNA binding fractions has been observed, and rechromatography of
‘specific’ DNA binding proteins fractions has yielded contradictory results.
Specific binding of non-histone
proteins to DNA probably occurs under a limited set of conditions, and
optimization of specific binding in vitro requires the determination of a
number of binding parameters. The nitrocellulose filter binding of
nucleoprotein complexes has provided a rapid and flexible DNA binding assay.
Several and co-workers39, 40, 81, for example, used tandem
heterologous and homologous DNA affinity chromatography to fractionate a rat
liver non-histone protein preparation, and then employed a filter binding assay
to analyze the binding specificities of the protein fractions obtained. At
saturation, the heterologous DNA binding proteins bound 60-70% of whole rat
liver DNA whereas the homologous DNA binding proteins boundonly 35% implying
the existance of some sequence specificity. Competition experiments
estabilished that the homologous DNA binding protein fraction bind homologous
DNA preferentially, with no such
preference being observed for the heterologous DNA binding protein fractions.
Analysis of the DNA retaind by filters showed that it is enriched four-fold
unique DNA sequences and depleted in repetitive sequence relative total DNA.
Cloned DNA sequences have also been
used to identify sequence-spesific binding. Hsieh and Brutlag36
isolated a protein from Drosophila
embryos which specifically binds to a cloned repeating unit of satellite DNA
when the plasmid and its insert are superhelical conformation. Retriction
endonuclease mapping of this DNA-protein complex revealed that the protein
protects one specific restriction site on the inserted DNA fragment.
Similiarly, Weideli et al.96
have isolated a DNA binding protein (DB-2) from Drosophila and bound it to random or retriction fragments of whole
cell DNA. Bound DNA fragments were recovered from nitrocellulose filters,
inserted into plasmids, and cloned in bacteria by standard recombinant DNA
procedures. The cloned DNA fragments were reacted with the DNA binding protein
again, and bound DNA fragments were cloned a second time. This process yielded
a cloned DNA fragment which selectively binds to the DB-2 protein at high ionic
strength (2.0 mol/ℓ NaCl). Immunocytochemical localization of the DB-2 protein
revealed that is concentrated specifically on chromosome 3 (section 95a/b) in
intact cells, supporting the postulate that the protein is associated with a
specific region of the Drosophila
genome in vivo.
One obvious disandventage to
experiments such as those described above, in which one monitors the binding of
proteins to purified DNA sequence, is that in vivo the DNA is part of chromatin
complex which may behave quite differently from naked DNA. A model system which
attempts to overcome this difficulty has been devised by Chao et. al.13 who used 1 203 base
pair DNA fragment containing the E.coli
lac operators sequence to reconstitute nucleosomal structures with call thymus
histone. When such nucleosomal were bound to the lac repressor protein,
sequence-spesific recognition could be detected without with the diplacement of
histone. Since the binding was quantitative and spesific, the results suggest
that the reconstitution protocol yields structure where the bound DNA strand
faces the outer surface of the nucleo-histone complex. Chemical crosslinking of
the histone inhibits binding of the lac repressor only occurs when the operator
sequence is positioned in the region of nucleosome stabilized by H2A-H2B
interaction (but not H3-H4 interactions). This implies that squence-spesific
binding in eukaryotic chromatin may be modulated by DNA interactions with
histone proteins which define the accessibility of sequence to intermoleculer
interactions.
Histone may not be the only
chromatin which influences the binding of non-histone proteins to DNA. Dastugue
and Crepin19 have reported that the binding of non-histone proteins
to isolated DNA exhibits less species specifity than the binding of the same
proteins to intact chromatin. This observation suggests that the spesific
binding of non-histone proteins to DNA may be influenced by chromatin proteins
other than histone histones, since the histones do not vary much from species
to species. Although the physicological significance, if any, of the in vitro
binding of non-histone proteins to chromatin is not certain, it is interesting
to note that such binding is accompanied by stimulation of transcription in
homologous, but not heterologous, protein/chromatin structure mixtures18.
An alternative approach to the
identification of the spesific DNA : non-histone protein complexes formed in
vivo has been provided by Bekhor and coworkers6, 7, 23, 66.
Essentially, chromatin is extensively washed with 2.0 mol/ℓ NaClto remove
loosely bound proteins, and the protein-depleted DNA is then dialysed against
10 mmol/ℓ Tris-HCl. Low speed centrifugation yields as nucleoprotein complex
containing some 1 % of the total nuclear DNA complexed to a spesific
non-histone protein fraction. The DNA in this complex is enriched twenty-fold
in transcribed sequences, and is depleted of non-transcribed sequences,
inactive gene sequences, and highly repentitive DNA sequences. Since the
proteins bind spesific DNA sequences with such high affinity, rearragement
during isolation is an unlikely explanation for this nucleoprotein complex.
