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J Biol Chem, Vol. 274, Issue 29, 20116-20122, July 16, 1999

Constitutive Phosphorylation of the Acidic Tails of the High Mobility Group 1 Proteins by Casein Kinase II Alters Their Conformation, Stability, and DNA Binding Specificity^*

Jacek R. Winiewski^§, Zbigniew Szewczuk^¶, Inga Petry^¶, Ralf Schwanbeck, and Ute Renner

From the  III. Zoologisches Institut-Entwicklungsbiologie, Universität Göttingen, Humboldtallee 34A, D-37073 Göttingen, Germany and the ^¶ Wydzia Chemii, Uniwersytet Wrocawski, ulica F. Joliot-Curie 14, PL-50383 Wrocaw, Poland

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The high mobility group (HMG) 1 and 2 proteins are the most abundant non-histone components of chromosomes. Here, we report that essentially the entire pool of HMG1 proteins in Drosophila embryos and Chironomus cultured cells is phosphorylated at multiple serine residues located within acidic tails of these proteins. The phosphorylation sites match the consensus phosphorylation site of casein kinase II. Electrospray ionization mass spectroscopic analyses revealed that Drosophila HMGD and Chironomus HMG1a and HMG1b are double-phosphorylated and that Drosophila HMGZ is triple-phosphorylated. The importance of this post-translational modification was studied by comparing some properties of the native and in vitro dephosphorylated proteins. It was found that dephosphorylation affects the conformation of the proteins and decreases their conformational and metabolic stability. Moreover, it weakens binding of the proteins to four-way junction DNA by 2 orders of magnitude, whereas the strength of binding to linear DNA remains unchanged. Based on these observations, we propose that the detected phosphorylation is important for the proper function and turnover rates of these proteins. As the occurrence of acidic tails containing canonical casein kinase II phosphorylation sites is common to diverse HMG and other chromosomal proteins, our results are probably of general significance.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

High mobility group (HMG)¹ proteins are abundant components of chromatin. A subfamily of the HMG proteins containing the HMG1 box domain (HMG1-BD) is widely distributed in eukaryotic cells from yeast to man (for a review, see Ref. 1). The members of this group are thought to have various functions related to modulation of transcription, DNA integration, and recombination. Since these proteins have an ability to induce strong bends and unwind DNA, they are called architectural components of chromatin. The most abundant of the HMG1 box proteins are the HMG1 and HMG2 proteins. They are composed of one or two HMG1-BDs, which are primarily responsible for contacts with DNA. HMG1-BDs are amino- and/or carboxyl-terminally flanked by stretches of positively or negatively charged residues. These regions modulate the binding affinity of HMG1-BDs (2-7), but do not influence the extent of DNA distortion (8). Deletion of these regions, in particular those of the negatively charged carboxyl-terminal tails, alters binding specificity of the HMG1 proteins (5). Moreover, the C-terminal portion of the HMG1 proteins is important for stimulation of transcription (9) and nuclear retention (10).

Two abundantly expressed proteins of this family were found in each of the dipteran insects Chironomus (cHMG1a and cHMG1b (11)) and Drosophila (HMGD (12) and HMGZ (13)). They are composed of a single HMG1-BD that is C-terminally flanked by a positively and a negatively charged region (for a review, see Ref. 14). Pulse-labeling studies on phosphorylation of the Chironomus proteins showed that they are phosphorylated within their positively charged regions by protein kinase C (15). Phosphorylation of the Chironomus HMG1 proteins by protein kinase C reduces the strength of their binding to DNA and affects their nucleocytoplasmic distribution (15). In mammals, mouse testis-specific HMG1 protein was found to be phosphorylated by protein kinase C (16). This modification appears to be required for both DNA binding and the topoisomerase I-dependent supercoiling activities of testis-specific HMG (16).

Here, we report that in addition to this regulatory phosphorylation by protein kinase C, HMG1 proteins in insects are constitutively phosphorylated by casein kinase II within their acidic C-terminal tails. This 2- or 3-fold modification alters the conformation, stability, and DNA binding properties of these proteins and therefore appears to be essential for their function. Our results are probably of general importance because putative CKII phosphorylation sites occur in acidic stretches of HMG proteins and other chromosomal proteins

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Protein Extraction-- The Drosophila HMGD and HMGZ proteins were isolated from 0-18-h embryos of an Oregon R strain. The embryos were collected for the desired period on apple juice-agar plates. They were washed off with water, thoroughly rinsed over a nylon filter, and stored at 80 °C. The frozen embryos were ground together with solid carbon dioxide in a laboratory grinder and thawed after addition of 5% (v/v) HClO₄. The resulting suspension was centrifuged at 10,000 × g for 10 min, and the proteins were precipitated from the supernatant with 33% Cl₃CCOOH at 0 °C for 1 h. The precipitate was washed with 0.2% HCl in acetone and subsequently two times with pure acetone. The pellets were dried under vacuum and stored at 20 °C. The Chironomus HMG1a and HMG1b proteins were isolated from cultured cells by extraction with 5% (v/v) HClO₄ in three freezing-thawing cycles (3). The cell supernatants were acidified with HCl to 0.35 M, precipitated with 6 volumes of acetone, and dried.

Protein Purification-- Crude extracts were separated on a reverse-phase C₁₈ Zorbax SB-300 column using an H₃CCN linear gradient in 0.1% F₃CCOOH/H₂O as described previously (3). The isolated proteins were rechromatographed on a second column of the same type and lyophilized.

Dephosphorylation of the Native Protein-- 100 µg of cHMG1a were incubated with 10 units of calf intestine alkaline phosphatase (Calbiochem) in 0.2 M Tris-HCl (pH 9.6) containing 10 mM MgCl₂ and 1 mM ZnCl₂ at 37 °C for 2-3 h. The dephosphorylated protein was purified by HPLC as described above. The dephosphorylated protein was free from native or partially dephosphorylated forms as judged by gel electrophoresis and mass spectroscopy.

Digestion of Proteins and Peptide Separation-- The proteins were digested with proteinase Glu-C in 25 mM NH₄HCO₃ at 20 °C for 24 h. The mass ratio of protein to proteinase was 20:1. Tryptic digestion of the C-terminal peptides was carried out in 0.1 M Tris-HCl (pH 7.5) at 37 °C for 3 h. The peptides were resolved on a reverse-phase C₁₈ Zorbax SB-300 column using an H₃CCN linear gradient in 0.1% F₃CCOOH/H₂O as described previously (15, 17).

Mass Spectra-- Mass spectra were recorded on a Finnigan MAT TSQ 700 triple-stage quadrupole mass spectrometer equipped with an electrospray ion source. Samples were typically dissolved in methanol/water/acetic acid (47:48:5, v/v/v) solution at a concentration of 50 pmol/µl and introduced into the electrospray needle by mechanical infusion through a microsyringe at a flow rate of 1 µl/min. A potential difference of 4.5 kV was applied between the electrospray needle. Nitrogen gas was used to evaporate the solvent from the charged droplets. At least 20 scans were averaged to obtain each spectrum. Transformations of the resulted spectra were performed using the BioWorks software package (Finnigan MAT).

Fluorescence Spectroscopy-- Fluorescence measurements were carried out on a Kontron SFM-25 spectrofluorometer using 10-nm slits for excitation and emission, and the fluorescence values were registered every 2 nm. The buffer used was 50 mM NaCl and 10 mM sodium phosphate (pH 6.9), and the protein concentration was 5 µM. The temperature dependence of the fluorescence intensity was corrected using L-tryptophan as a standard. The fluorescence intensity values were transformed into fraction of unfolded protein (f_u) molecules and used to calculate the free energies of unfolding using the relationship shown in Equation 1,

(Eq. 1)

where K_u is the unfolding constant, R is the gas constant, and T is the temperature. The melting temperature (T_m) was at G = 0 (18).

Derivative UV Spectroscopy-- The protein spectra were recorded and processed on a Kontron Uvikon 940 UV-visible spectrophotometer in 50 mM NaCl and 10 mM sodium phosphate (pH 6.9). The slit with was 2 nm; the scanning speed was 20 nm/min; and the absorption was registered every 0.1 nm. The measurements were done at 20 °C. The protein concentration was 27 µM. The relative tyrosine unfolding was calculated according to Equation 2,

(Eq. 2)

where _dp and _n are the fractional exposures of tyrosine in dephosphorylated and native proteins, respectively. The _dp and _n values were estimated from their second-derivative spectra according to Equations 3 and 4,

(Eq. 3)

(Eq. 4)

where R_n, R_dp, and R_u are numerical values related to changes in the tyrosyl microenvironments (19) on the native, dephosphorylated, and fully unfolded protein, respectively. R_a is the value obtained for the mixture of N-acetyl-Tyr-NH₂ and N-acetyl-Trp-NH₂ dissolved in ethylene glycol. The molar ratio of the amino acid derivatives was 2:3, as it has the cHMG1a protein. The R values were calculated from Equation 5,

(Eq. 5)

where A" is the second-derivative absorbance at 283, 287, 290.5, and 295 nm (19).

Limited Proteolytic Digestion-- A mixture of native and dephosphorylated cHMG1a proteins (1:1 molar ratio) was digested with chymotrypsin (treated with N-tosyl-L-lysine chloromethane), thermolysin, or trypsin in 30 mM NaCl and 25 mM Tris-HCl (pH 7.5) at 20 °C. The ratio of protein to enzyme was 50:1 (w/w). The reactions were terminated by mixing with an equal volume of 10 M urea solution containing 5% (v/v) acetic acid, 4% (v/v) 2-mercaptoethanol, 10 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, and 0.2 mM N-tosyl-L-lysine chloromethane. The reaction products were separated on urea-acetic acid-Triton X-100-15% polyacrylamide gels (20).

Mobility Shift Assay-- The ³²P-labeled four-way junction DNA and AT-rich Chironomus satellite DNA were prepared as described previously (3, 21). Briefly, the proteins were incubated together with labeled DNA in 80 mM NaCl, 1 mM MgCl₂, 0.01% bovine serum albumin, 8% glycerol, and 10 mM Tris-HCl (pH 7.9) at 20 °C for 10 min. The complexes of proteins with DNA were run on 6% polyacrylamide gels containing 2.5% (v/v) glycerol, 6.75 mM Tris-HCl, 3.3 mM sodium acetate, and 1 mM EDTA (pH 7.9). The gels were dried and autoradiographed.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

HMGD, cHMG1a, and cHMG1b Proteins Are Phosphorylated at Two Positions-- The mass spectra of the entire HMGD, cHMG1a, and cHMG1b proteins revealed that each protein is twice phosphorylated and carries a single acetyl group because their M_r values were ~202 higher than the M_r values calculated from their sequences. Relatively smaller portions of the proteins were found to be monophosphorylated, and a negligible amount possesses no phosphoryl group at all. Deacetylated species were not detected. A typical example of a transformed spectrum of the native cHMG1a protein is shown in Fig. 1A. The M_r of 13,116 corresponds to acetylated and double-phosphorylated cHMG1a. Two other signals with M_r values 13,036 and 12,956 differ by 80 and 160 units, respectively. The spectra of the other HMG proteins show a similar pattern, although their M_r values differ from that of cHMG1a. To confirm that the 80-unit shift is due to phosphorylation, the native proteins were treated with alkaline phosphatase. Fig. 1B shows that the enzyme treatment reduced the molecular weight of the phosphoprotein back to that of acetylated cHMG1a (M_r 12,956).

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Electrospray ionization-transformed spectra obtained for native cHMG1a (A) and its dephosphorylated product (B). The spectrum in A shows the presence of three components with Mr values of 12,956, 13,036, and 13,116, differing by 80 units. Only one main peak with a Mr of 12,956 is present in the spectrum in B. The Mr value of 12,956 is in agreement with the calculated Mr of acetylated cHMG1a.

Phosphorylation Sites Are Located within C-terminal Tails and Match the Consensus Substrate Sites for Casein Kinase II-- To obtain more detailed information about the location of phosphorylation and acetylation sites in the proteins, cHMG1a, cHMG1b, and the mixture of HMGD and HMGZ (Table I) were digested with proteinase Glu-C. The cleavage products were separated by HPLC (data not shown), and the obtained fractions were analyzed by electrospray ionization mass spectrometry. The molecular weights of peptides 13, 20, and 28 (Table I) indicate that the N termini of cHMG1a, cHMG1b, and HMGD are acetylated. The analogous N-terminal segment of HMGZ has not been detected. The spectra revealed that cHMG1a, cHMG1b, and HMGD were phosphorylated twice within their C-terminal peptide, whereas three phosphate groups were localized in the C-terminal peptide of HMGZ (Table I).

The molecular weight of the C-terminal peptide of cHMG1a (peptide 3; M_r 2853.8) corresponds to the molecular weight calculated from its sequence. This indicates that there are two phosphorylations in this fragment. The peptide includes three serines, making it difficult to identify the phosphorylation sites. Subdigestion of the peptide with trypsin resulted in the peptide (MH⁺ 1572) that corresponds to the double-phosphorylated fragment 102-113, containing just two serine residues (data not shown). This result clearly places the phosphorylation sites at Ser-103 and Ser-112 (Table II) and is in agreement with the postulated single phosphorylation of peptide 5 (fragment 110-113), containing one serine only.

The C-terminal peptide 21 of HMGZ (fragment 103-110) contains only one site of phosphorylation, allowing us to map the phosphorylation position to Ser-109 (Table II). All mapped phosphorylation sites match consensus substrate sites for CKII (22, 23). Since Ser-100 and Ser-101 in HMGZ, Ser-102 and Ser-108 in cHMG1b, and Ser-102 and Ser-110 in HMGD also match the phosphorylation sites for CKII, it is very likely that these sites are phosphorylated in these proteins (Table II). Furthermore, we found that in vitro, the cHMG1a protein is efficiently phosphorylated by human CKII (data not shown). This supports additionally the possibility that insect CKII is responsible for phosphorylation of the proteins. To obtain insight in the biological meaning of the observed constitutive phosphorylation of the HMG1 proteins, we compared the biophysical and biochemical properties of the native (phosphorylated) and alkaline phosphatase-dephosphorylated proteins.

Phosphorylation Alters Protein Conformation and Thermal Stability-- The HMG1-BDs of the insect proteins contain three tryptophanyl residues. In previous fluorescence studies using recombinant cHMG1a proteins, we showed that one of these residues, Trp-14, is exposed to solvent (3). The maximum of the fluorescence emission of this residue is 350 nm. In contrast, the two other Trp residues are buried in the protein interior and exhibit a maximum of fluorescence at 320 nm. In addition, we found that deletion of the acidic tail of the cHMG1a protein results in an increase in fluorescence intensity, suggesting that the C-terminal part of the protein quenches or alters the environment of tryptophan residues (3). Since double phosphorylation within the acidic tail of the protein might contribute to a specific conformation, we compared the Trp fluorescence spectra of the native and dephosphorylated cHMG1 proteins (Fig. 2A). The emission spectra of the tryptophans of the native and dephosphorylated proteins exhibited fluorescence maxima at 329 and 337 nm, respectively. Furthermore, a measurable increase in fluorescence intensity was observed as a result of the protein dephosphorylation. The observed red shift of 8 nm suggests strong changes in protein conformation that might mainly involve the spatial arrangement of the C terminus in respect to the HMG1-BD.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Fluorescence spectroscopy of native and dephosphorylated cHMG1a protein. The excitation wavelength was 295 nm. A, emission spectra at 20 °C; B and C, thermal denaturation. circles, native cHMG1a; squares, dephosphorylated cHMG1a; triangles, fluorescence of L-tryptophan used as a standard. The fluorescence intensity was measured at 350 nm.

In thermal denaturation experiments, we observed that the native protein exhibits a higher melting temperature (T_m = 46.3 °C) than the dephosphorylated protein (T_m = 43.9 °C) (Fig. 2, B and C). The difference in the transition temperatures of 2.4 °C shows that the phosphates importantly contribute to the protein stability. The dephosphorylation of the protein leads to a substantial reduction of the free energy of unfolding (G_u). At 20 °C the G_u values for native and dephosphorylated proteins were 13.6 and 12.8 kJ/mol, respectively (Fig. 2C, inset). These relatively low values are in good agreement with previously reported moderate conformational stability of the cHMG1a protein (24).

Second-derivative near-UV absorption spectroscopy is a useful tool for examining the state of tyrosyl residues in proteins also containing tryptophan (25). We used this technique to analyze the changes upon protein dephosphorylation in the microenvironments of tyrosyl residues in cHMG1. Fig. 3 shows the second-derivative spectra of native and dephosphorylated proteins. A value of relative change upon protein dephosphorylation of the solvent exposition of tyrosyl residues was calculated. The value Y_u = 2.1 suggests a 2-fold increase in tyrosyl residue exposition in the dephosphorylated protein compared with its native form. This result is in good agreement with the spectral properties of both forms observed in fluorescence emission spectra. Since Tyr-11 is located adjacent to Trp-14, it is likely that perturbations in the tyrosine component are mainly due to changes within the microenvironment of Tyr-11.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Second-derivative UV absorption spectra of native and dephosphorylated cHMG1a proteins. The Rn, Rdp, and Ra values of 0.56, 0.66, and 0.47 were calculated from the spectra of the native protein (· · · ·), dephosphorylated protein (- - -), and a mixture of N-acetyl-Tyr-NH2 and N-acetyl-Trp-NH2 (- · -), respectively. The model compounds were dissolved in ethylene glycol, a solvent possessing characteristics of the interior of the protein matrix (19, 25).

Phosphorylation Stabilizes the Protein against Digestion of Some Proteinases-- Proteolytic enzymes are useful tools in the detection and characterization of changes in the tertiary structure of proteins. The mixture of native and dephosphorylated cHMG1a proteins was partially digested by chymotrypsin, thermolysin, and trypsin (Fig. 4). In the presence of chymotrypsin and thermolysin, the dephosphorylated protein was digested more rapidly than the native protein (Fig. 4, B, C, and E). Chymotrypsin specifically hydrolyzes peptide bonds at hydrophobic residues, whereas thermolysin does it preferentially; therefore, these data suggest an increased exposition of apolar residues upon dephosphorylation. These results confirm our spectroscopic data showing an increased exposition of tryptophanyl and tyrosyl residues in the dephosphorylated cHMG1a protein. In contrast, trypsin, which specifically cuts peptide bonds at carboxyl termini of arginyl and lysinyl residues, digested the native protein form more rapidly (Fig. 4, D and E). Thus, it is likely that the accessibility of the basic residues to trypsin also changes upon protein phosphorylation.

View larger version (29K): [in this window] [in a new window]   Fig. 4.   Time course of digestion of native and dephosphorylated cHMG1a proteins by chymotrypsin, thermolysin, and trypsin. A, undigested protein mixture. The arrows indicate the positions of the intact forms of the protein (n, native; dp, dephosphorylated). The asterisks indicate the position of the more stable form. The open circle indicates the position of fragment 1-84, which comprises the HMG1-BD of cHMG1a. The protein bands in A, the 120 min lane in B and C, and the 4 min lane in D were scanned and quantified. The ratios of the remaining dephosphorylated and native forms (dp:n) are shown in E. Contr., control; Therm., thermolysin; Chym., chymotrypsin; Tryp., trypsin.

Phosphorylation Affects DNA Binding Properties of the Proteins-- HMG1-BD proteins bind preferentially to the DNAs in a non-B conformation. This includes intrinsically prebent, cruciform, bulged, and cis-platinated DNAs. Previously, we have demonstrated that the acidic tail inhibits the binding affinity of the protein for linear and four-way junction DNAs (3) and that phosphorylation at protein kinase C sites additionally weakens the interaction of cHMG1a and cHMG1b with DNA (15). More recently, Payet and Travers (5) demonstrated that the presence of the acidic tail in the recombinant HMGD protein is essential for its structure-specific recognition of such DNAs. Because the phosphorylation might contribute to protein specificity, we compared the binding properties of the native and dephosphorylated cHMG1a proteins using four-way junction and linear AT-rich DNAs, which possess multiple binding sites. The native proteins produced two shifts with the four-way junction DNA. The one with the higher mobility reflects interaction with the central portion of the junction (Fig. 5C, arrowhead), whereas the second more slowly migrating complex (arrow) corresponds to protein binding to the arm of the junction (26). Both protein-DNA complexes appeared for the first time at protein concentrations in the range of 30-100 nM. This suggests a similar binding affinity of the protein for both duplex arm(s) and the junction. The protein dephosphorylation essentially increased the binding affinity of the protein for the junction (Fig. 5D). At 1 nM protein, essentially the entire DNA was bound. Thus, an ~2 orders of magnitude increase in protein affinity for the junction was found, but not for the arm. In experiments in which the binding of both proteins to linear DNA was compared, apparently no difference in the binding affinity was found (Fig. 5, A and B); however, an altered binding specificity was observed. The native protein was able to bind at different sites of the DNA, whereas the dephosphorylated protein preferentially bind to a single site on the DNA.

View larger version (96K): [in this window] [in a new window]   Fig. 5.   Binding of native (A and C) and dephosphorylated (B and D) cHMG1a proteins to double-stranded (A and B) and four-way junction (C and D) DNAs. 32P-Labeled ClaI-DNA fragment or four-way junction DNA (<0.1 nM) was incubated with increasing concentrations of the proteins and electrophoresed on 6% polyacrylamide gels. F, free DNA. The gels were dried and autoradiographed. The arrows indicate bands that distinguished shifts caused by the native protein, but not by the dephosphorylated protein. The arrowheads show the positions of the shifts specific to both protein forms.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Our results show that Drosophila and Chironomus HMG1 proteins are constitutively phosphorylated within their C-terminal tails by CKII. This phosphorylation is important for their proper folding, thermodynamic and metabolic stability, and DNA binding specificity.

CKII is a structurally and functionally conserved enzyme that is widely distributed among eukaryotic organisms (22). However, the biological role of this kinase is only poorly understood; it appears to be involved in the modulation of properties of some transcription factors and as well as in the regulation of cell proliferation (23). Furthermore, the enzyme is essential for viability of Saccharomyces cerevisiae (27). This stresses the importance of this kinase in eukaryotic cells.

In Drosophila and Chironomus cells, almost the entire population of HMG1 proteins was found to be double- or triple (HMGZ)-phosphorylated. Since only small amounts of partially dephosphorylated species were detected, it appears that the modification of the acidic tails of the HMG proteins is important for their proper function. The extent of phosphorylation of Drosophila HMG1 proteins remains constant during the entire development²; and therefore, it is likely that the modification of these proteins by CKII is constitutive. The phosphorylation of the tails changes the DNA binding properties of these proteins with respect to their structure specificity.

The phosphorylation of chromosomal proteins by CKII appears to be a common property found in evolutionarily distant organisms. In plant HMG proteins (28) and Drosophila protein D1 (similar to HMGI/Y) (29), substrates for CKII isolated from these organisms were found. Members of the HMGI/Y family were found to be phosphorylated in vivo by CKII within their acidic C-terminal tails (Table II) (30, 31). Multiple phosphorylation sites were found in Drosophila heterochomatin-associated protein (HP-1) (32). Inspection of the primary structure of this protein reveals at least two possible CKII phosphorylation sites (Table II). Multiple putative CKII sites are also present in the acidic tails of structurally related HP-1 proteins in mammals (protein M31). Moreover, HMG1 box-containing, structure-specific recognition proteins, upstream binding factor, and plant HMG1 proteins possess long acidic tails with canonical phosphorylation sites of CKII (Table II).

What might be the functional significance of phosphorylation of HMG and other chromosomal proteins by CKII? The levels of the HMG proteins are variable between different types of cells (33). Usually undifferentiated and rapidly proliferating cells contain higher amounts of these proteins compared with terminally differentiated cells. However, the biological meaning of these differences as well as the mechanisms regulating the titers of the HMG proteins are not clearly understood. In the insect systems of Drosophila and Chironomus, HMG proteins in vivo are metabolically relatively stable, and their turnover rates extend over many cell generations (34). The entire population of HMG1 proteins is CKII-phosphorylated, suggesting that intermediate or dephosphorylated forms are only short-living. The results presented show that dephosphorylation of cHMG1a causes a partial denaturation and reduction of the stability of the protein against proteinase in vitro. Taking these facts together, we suggest that CKII phosphorylation is essential for the metabolic stability of these HMG1 proteins in vivo; the dephosphorylation of these proteins might be a part of the mechanism regulating their titer, in particular, during cell differentiation.

However, the phosphorylation of the acidic tails of the HMG proteins seems to be widely distributed; some groups of these proteins appear to be not modified by CKII. The HMG1 and HMG2 proteins containing two HMG1-BDs from vertebrate organisms, Drosophila DSP-1 (dorsal-switch protein 1), and yeast ACP2 (acidic protein 2) do not possess canonical CKII phosphorylation sites in their C-terminal acidic tails. In the HMG14/17 family, only the HMG14 protein is a substrate for CKII, whereas the HMG17 protein is not. Despite that fact that these proteins are very similar in their primary structures, they were localized to distinct regions of chromatin (35). This selectivity might be due to phosphorylation of the acidic tail of HMG14.

Recombinant technology (and in particular, the possibility of producing eukaryotic proteins in bacteria) has revolutionized biochemistry. However, many of these proteins, such as those described in this work, are post-translationally modified in eukaryotic cells. Unfortunately, in bacteria, these proteins are not phosphorylated and acetylated. Because these modifications are important, such proteins should be phosphorylated in vitro prior to biochemical analyses. This is easily to perform² since CKII preparations are commercially available. In many proteins, the CKII sites are located several residues from C termini. This offers the possibility of end labeling such proteins. Their conformation and interaction with DNA could be analyzed by the protein footprinting method without the introduction of artificial phosphorylation sites (36).

The data presented in this work demonstrate the importance of the constitutive phosphorylation of a group of HMG proteins. This modification appears to be essential for the function of these proteins. Further modifications of HMG proteins, including phosphorylation by protein kinase C (15, 16), Cdc2 kinase (17, 37, 38), and mitogen-activated protein kinase (17) and ADP-ribosylation (41), facultatively change fractions of these proteins at particular events of cell life, such as mitosis, differentiation, and apoptosis.

	ACKNOWLEDGEMENTS

We thank Dr. J. Ziókowski (University of Wrocaw) and Dr. U. Grossbach (University of Göttingen) for the interest in and the support of this work. Dr. M. A. Schäfer (University of Göttingen) is gratefully acknowledged for the continuous supply of Drosophila flies.

	FOOTNOTES

^* This work was supported by Deutsche Forschungsgemeinschaft Grant Wi-1210/2-1 (to J. R. W.) and Komitet Bada Naukowych Grant 4P05A 023 14 (to Z. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence should be addressed. Fax: 49-551-395416; E-mail: jwisnie@gwdg.de.

² J. R. Winiewski and U. Renner, unpublished results.

	ABBREVIATIONS

The abbreviations used are: HMG, high mobility group; HMG1-BD, HMG1 DNA-binding domain; cHMG1, Chironomus HMG1; HMGD, Drosophila HMG protein D; HMGZ, Drosophila HMG protein Z; CKII, casein kinase II; HPLC, high pressure liquid chromatography.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

1.	Bustin, M., and Reeves, R. (1996) Prog. Nucleic Acids Res. Mol. Biol. 54, 35-100[Medline] [Order article via Infotrieve]
2.	Sheflin, L. G., Fucile, N. W., and Spaulding, S. W. (1993) Biochemistry 32, 3228-3248
3.	Winiewski, J. R., and Schulze, E. (1994) J. Biol. Chem. 269, 10713-10719[Abstract/Free Full Text]
4.	tros, M., tokrová, J., and Thomas, J. O. (1994) Nucleic Acids Res. 22, 1044-1051[Abstract/Free Full Text]
5.	Payet, D., and Travers, A. (1997) J. Mol. Biol. 266, 66-75[CrossRef][Medline] [Order article via Infotrieve]
6.	Ritt, C., Grimm, R., Fernández, S., Alonso, J. C., and Grasser, K. D. (1998) Biochemistry 37, 2673-2681[CrossRef][Medline] [Order article via Infotrieve]
7.	tros, M. (1998) J. Biol. Chem. 273, 10355-10361[Abstract/Free Full Text]
8.	Heyduk, E., Heyduk, T., Claus, P., and Winiewski, J. R. (1997) J. Biol. Chem. 272, 19763-19770[Abstract/Free Full Text]
9.	Aizawa, S., Nishino, H., Saito, K., Kimura, K., Shirakawa, H., and Yoshida, M. (1994) Biochemistry 33, 14690-14695[CrossRef][Medline] [Order article via Infotrieve]
10.	Shirakawa, H., Tanigawa, T., Sugiyama, S., Kobayashi, M., Terashima, T., Yoshida, K., Arai, T., and Yoshida, M. (1997) Biochemistry 36, 5992-5999[CrossRef][Medline] [Order article via Infotrieve]
11.	Winiewski, J. R., and Schulze, E. (1992) J. Biol. Chem. 267, 17170-17177[Abstract/Free Full Text]
12.	Wagner, C. R., Hamana, K., and Elgin, S. C. R. (1992) Mol. Cell. Biol. 12, 1915-1930[Abstract/Free Full Text]
13.	Ner, S. S., Churchill, M. E. A., Searles, M. A., and Travers, A. A. (1993) Nucleic Acids Res. 21, 4369-4371[Abstract/Free Full Text]
14.	Winiewski, J. R. (1998) Zool. Pol. 43, 5-24
15.	Winiewski, J. R., Schulze, E., and Sapetto, B. (1994) Eur. J. Biochem. 255, 687-693
16.	Alami-Ouahabi, N., Veilleux, S., Meistrich, M. L., and Boissonneault, G. (1996) Mol. Cell. Biol. 16, 3720-3729[Abstract]
17.	Schwanbeck, R., and Winiewski, J. R. (1997) J. Biol. Chem. 272, 27476-27483[Abstract/Free Full Text]
18.	Peace, C. N., Shirley, B. A., and Thomson, J. A. (1989) in Protein Structure: A Practical Approach (Creighton, T. E., ed) , pp. 311-330, IRL Press, Oxford
19.	Ragone, R., Colona, G., Belestrieri, C., Servillio, L., and Irace, C. (1984) Biochemistry 23, 1871-1875[CrossRef][Medline] [Order article via Infotrieve]
20.	Zweidler, A. (1978) Methods Cell Biol. 17, 223-233[Medline] [Order article via Infotrieve]
21.	Winiewski, J. R., Ghidelli, S., and Steuernagel, A. (1994) J. Biol. Chem. 269, 29261-29164[Abstract/Free Full Text]
22.	Glover, C. V. C. (1995) in The Protein Kinase Facts Book: Protein-Serine Kinases (Hardie, G. , and Hanks, S., eds) , pp. 243-248, Academic Press Ltd., London
23.	Meisner, H., and Czech, M. P. (1995) in The Protein Kinase Facts Book: Protein-Serine Kinases (Hardie, G. , and Hanks, S., eds) , pp. 240-242, Academic Press Ltd., London
24.	Winiewski, J. R., Heßler, K., Claus, P., and Zechel, K. (1997) Eur. J. Biochem. 243, 151-159[Medline] [Order article via Infotrieve]
25.	Havel, H. A. (ed) (1996) Spectroscopic Methods for Determining Protein Structure in Solution , pp. 62-68, VCH Publishers, Inc., New York
26.	Hill, A. D., and Reeves, R. (1997) Nucleic Acids Res. 25, 3523-3531[Abstract/Free Full Text]
27.	Padmanabha, R., Chen-Wu, J. L., Hanna, D. E., and Glover, C. V. C. (1990) Mol. Cell. Biol. 10, 4089-4099[Abstract/Free Full Text]
28.	Grasser, K. D., Maier, U.-G., and Feix, G. (1989) Biochem. Biophys. Res. Commun. 162, 256-261
29.	Glover, C. V. C., Shelton, E. R., and Brutlag, D. L. (1983) J. Biol. Chem. 258, 3258-3265[Abstract/Free Full Text]
30.	Palvimo, J., and Linnala-Kankkunen, A. (1989) FEBS Lett. 257, 101-104[CrossRef][Medline] [Order article via Infotrieve]
31.	Ferranti, P., Malorni, A., Marino, G., Pucci, P., Goodwin, G. H., Manfioletti, G., and Giancotti, V. (1992) J. Biol. Chem. 267, 22486-22489[Abstract/Free Full Text]
32.	Eissenberg, J. C., Ge, Y., and Harnett, T. (1994) J. Biol. Chem. 269, 21315-21321[Abstract/Free Full Text]
33.	Mosevitsky, M. L., Novitskaya, V. A., Iogannsen, M. G., and Zabezhinsky, M. A. (1989) Eur. J. Biochem. 185, 303-310[Medline] [Order article via Infotrieve]
34.	Ghidelli, S., Claus, P., Thies, G., and Winiewski, J. R. (1997) Chromosoma (Berl.) 105, 369-379[Medline] [Order article via Infotrieve]
35.	Postnikov, Y. V., Herrera, J. E., Hock, R., Scheer, U., and Bustin, M. (1997) J. Mol. Biol. 274, 454-465[CrossRef][Medline] [Order article via Infotrieve]
36.	Frank, O., Schwanbeck, R., and Winiewski, J. R. (1998) J. Biol. Chem. 273, 20015-20020[Abstract/Free Full Text]
37.	Reeves, R., Langan, T. A., and Nissen, M. S. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 1671-1675[Abstract/Free Full Text]
38.	Nissen, M. S., Langan, T. A., and Reeves, R. (1991) J. Biol. Chem. 266, 19945-19952[Abstract/Free Full Text]
39.	Hershey, J. C., Hautmann, M., Thompson, M. M., Rothblum, L. I., Haystead, T. A., and Owens, G. K. (1995) J. Biol. Chem. 270, 25096-25101[Abstract/Free Full Text]
40.	Bodenbach, L., Fauss, J., Robitzki, A., Krehan, A., Lorenz, P., Lozeman, F. J., and Pyerin, W. (1994) Eur. J. Biochem. 220, 263-267[Medline] [Order article via Infotrieve]
41.	Giancotti, V., Bandiera, A., Sindici, C., Perissin, L., and Crane-Robinson, C. (1996) Biochem. J. 317, 865-870
42.	Goodwin, G. (1998) Int. J. Biochem. Cell Biol. 30, 761-766[CrossRef][Medline] [Order article via Infotrieve]
43.	Walton, G. M., Spiess, J., and Gill, G. N. (1985) J. Biol. Chem. 260, 4745-4750[Abstract/Free Full Text]
44.	Kulikova, O. G., and Reikhardt, B. A. (1996) Biokhimiya 61, 1046-1054

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