Tioconazole The functions of HMGN protein are modulated by v

The functions of HMGN1 protein are modulated by various post-translational modifications, including phosphorylation , , , acetylation , and glycosylation . Phosphorylation in HMGN1 may influence its binding affinity to DNA or nucleosome-associated proteins and affect the sub-cellular localization and function of HMGN1 , , . In this regard, mitotic phosphorylation of HMGN1 has been shown to prevent its binding to chromatin , enhance its interaction with 14.3.3 proteins, and inhibit its reentry into the nascent nucleus in late telophase . Several serine residues in HMGN1 protein have been shown to be phosphorylated by protein kinases in vitro. In this respect, HMGN1 isolated from calf thymus can be phosphorylated in vitro by protein kinase C; Ser20 and Ser24 were proposed to be the potential major and minor phosphorylation sites . In addition, Ser6 and Ser24 were found to be the major and minor sites of phosphorylation catalyzed by cAMP-dependent protein kinase (A-kinase) and cGMP-dependent protein kinase (G-kinase) , , . Moreover, Ser89 and Ser99 in bovine HMGN1 were shown to be the major and minor recognition sites of protein kinase CK2 (previously known as casein kinase II) . In the present paper, we employed LC–MS/MS and examined the sites of in vitro phosphorylation of human HMGN1 protein catalyzed by recombinant human protein kinase CK2. Our results showed that five serine residues, i.e., Ser6, Ser7, Ser85, Ser88, and Ser98, in human HMGN1 can be phosphorylated by CK2, which paralleled our previous observation with the nature of phosphorylation of HMGN1 protein isolated from MCF-7 human breast cancer Tioconazole . Materials and methods Cell culture. The human breast mammary epithelial cell line MCF-7 (ATCC, Manassas, VA) was cultured in minimum essential medium supplemented with 10% fetal bovine serum (ATCC, Manassas, VA), 0.01mg/mL bovine insulin (Sigma–Aldrich, St. Louis, MO), 100IU/mL penicillin, and 100μg/mL streptomycin (ATCC, Manassas, VA). Protein extraction and purification. The HMG proteins were extracted with 5% perchloric acid (PCA) as previously described [3]. PCA-soluble proteins were purified on a Surveyor HPLC system (ThermoFinnigan, San Jose, CA) by using a 1.5×250mm C4 column (Varian, Walnut Creek, CA). The flow rate was 0.2mL/min, and a 75-min gradient of 5% to 30% CH3CN in 0.1% aqueous solution of trifluoroacetic acid (TFA) was employed. The chromatogram was recorded by absorbance detection at 220nm. The amount of HMGN1 protein was determined by Bradford micro-assay (Bio-Rad, Hercules, CA). Calf intestinal alkaline phosphatase (CIP) treatment. The dephosphorylation reaction was carried out by incubating 10μg HMGN1 protein with 5U of CIP (New England Biolabs, Beverly, MA) at 30°C for 30min in a buffer containing 50mM NH4HCO3. The dephosphorylated HMGN1 protein was again purified by HPLC with a 0.50×150mm C4 column (Vydac, Hesperia, CA) on an Agilent 1100 system (Agilent Technologies, Palo Alto, CA) equipped with a UV detector. The flow rate was 6.0μL/min, and a 75-min gradient of 0–30% acetonitrile in 0.1% TFA was employed. The sample was then dried and subjected to MALDI-TOF MS analysis or in vitro CK2 reaction. In vitro phosphorylation by protein kinase CK2. The phosphorylation reaction was carried out at 30°C for 6h by incubating ∼10-μg of the above dephosphorylated HMGN1 protein with 36,000U of protein kinase CK2 (New England Biolabs, Beverly, MA) in a 350-μL 1× CK2 buffer supplied by the vendor. After the reaction, a small aliquot was sampled and cleaned by C18 Zip Tip (Millipore, Billerica, MA) and subjected to mass spectrometric analysis. Trypsin digestion. Sequencing-grade modified trypsin was purchased from Roche Diagnostics (Penzberg, Germany). The digestion was carried out in a 50-μL of 100mM NH4HCO3 (pH 8.0) solution containing approximately 0.2μg HMGN1 and the trypsin-to-substrate ratio was approximately 1 to 50. The digestion was continued at 37°C for overnight, and the reaction was terminated by adding 1-μL TFA.