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Camp-Dependent Phosphorylation of Bovine Lens Cv-Crystallin*

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Camp-Dependent Phosphorylation of Bovine Lens Cv-Crystallin* Proc. Natl. Acad. Sci. USA Vol. 82, pp. 4712-4716, July 1985 Cell Biology cAMP-dependent phosphorylation of bovine lens cv-crystallin* (AX, A2, B1, and B2 chains/post-translational modification/protein kinase) ABRAHAM SPECTOR, RAJL CHIESA, JANET SREDYt, AND WILLIAM GARNER Biochemistry and Molecular Biology Laboratory, Department of Ophthalmology, College of Physicians and Surgeons, Columbia University, New York, NY 10032 Communicated by Karl Meyer, March 18, 1985 ABSTRACT This communication reports that the Al and A few of the major cAMP-dependent phosphorylated pro- B1 chains of bovine lens a-crystallin are phosphorylated. The teins found in the lens have been identified. They include the conclusion is based on the following evidence: (i) When soluble major fiber membrane protein MP26 (4, 5), vimentin (6), actin preparations from lens cortex are incubated with [y-32P]ATP, (6), and fodrin (7). a cAMP-dependent labeling of a high molecular weight protein Of particular interest is the preliminary finding of a major is obtained. (it) After NaDodSO4/PAGE, the label is found in phosphorylated polypeptide with Mr in the 20,000 range, two bands with Mr 22,000 and 20,000, corresponding to the B which appears in the outer fiber layers (the outer cortex) but and A chains of a-crystallin, respectively. (Wil) Isoelectric not in the epithelium (3). This report presents evidence which focusing indicates that the radioactivity is almost exclusively in suggests that this phosphorylated fraction consists of the Al bands with pI values of 5.58 and 6.70, corresponding to the Al and B1 chains ofa-crystallin, one of the major proteins of the and B1 chains, respectively. (iv) Similar results are obtained in fiber cell. The phosphorylation may be involved in the experiments of [32P]orthophosphate incorporation in lens or- process ofterminal differentiation and the organization ofthe gan culture. (v) Analyses of the digested protein indicate the fiber cell. Thus, the major lens proteins, previously thought label is exclusively in phosphoserine. (VI) 31P NMR analyses of to only contribute to the uniform refractive index of the native, proteolytically digested, and urea-treated a-crystallin tissue, may have other functions and be under stringent gives a chemical shift of 4.6 ppm relative to 85% H3P04 at pH metabolic control. 7.4, suggesting that the phosphate is covalently bound to a serine in the protein. An abundance of approximately one MATERIALS AND METHODS phosphate per four or five monomer units was found. (vii) Similar results were obtained by chemical analyses of indepen- A soluble fraction from calf lens outer cortex (LOCSF) was dently prepared a-crystallin samples. The results are consist- prepared with bovine eyes from approximately 15-week-old ent with the view that the Al and B1 chains arise as result of the calves obtained from a local slaughterhouse. The lenses phosphorylation of directly synthesized A2 and B2 polypep- (1.2-1.6 g) were removed from the eyes and decapsulated by tides. It is suggested that this metabolically controlled phos- dissection. Outer cortex fiber cells (10-20% by weight) were phorylation may be associated with the terminal differentiation obtained by placing the decapsulated lenses in a buffer of the lens epithelial cell and the intracellular organization of containing 10 mM Hepes, and 50 mM 2-mercaptoethanol, pH the lens fiber cell. 7.4, and stirring vigorously for 5-10 min on ice. The suspen- sion of fiber cells was homogenized by using a Because protein phosphorylation is an important reaction Potter-Elvehjem homogenizer and centrifuged at 105,000 x involved in the control of a number of diverse biological g for 45 min. The supernatant, containing 30-50 mg ofprotein processes such as cell differentiation and gene expression (1), per ml, was collected, divided into aliquots, quick frozen investigation of phosphorylation reactions in the lens of the under liquid nitrogen, and stored at -800C until used. eye was initiated. The lens is a particularly inviting tissue for In vitro phosphorylation of the LOCSF preparation was such study. It contains a single layer of epithelial cells, which performed in 200 ,ul of reaction mixture containing 20 mM in the equatorial region are constantly going into terminal imidazole, 1 mM MgCl2, 0.02 mM cAMP, 4 mM NaF, 2 mM differentiation (2). In this process, the cells increase dramat- theophylline, 0.3 tLM [y-32P]ATP (3000 Ci mmol-1, obtained ically in volume, extending to both the anterior and posterior from New England Nuclear; 1 Ci = 37 GBq), and 4 mg of side of the tissue. The cells lose their nuclei and gradually protein. The reaction, started by the addition of [y32P]ATP, their ability to synthesize protein. During terminal differen- was carried out at 370C for 10 min and stopped by addition of tiation, an entirely new population of cytosol and membrane ice-cooled trichloroacetic acid to bring a final concentration proteins is produced. The new fibers are constantly displac- of 10%. The protein precipitate was collected by centrifuga- ing older fibers in towards the center of the tissue. This tion at 12,000 x g for 15 min and washed five times with 95% process results in the production of an age-dependent gradi- (vol/vol) ethanol and five more times with diethyl ether. In ent with the oldest, metabolically inactive cells in the center certain experiments, the phosphorylation reaction was ofthe tissue and the youngest fibers in the outer perimeter of stopped by injection of the incubation mixture in an HPLC the tissue in contact with the epithelium. system to be immediately fractionated. Previous studies on protein phosphorylation in the lens (3) [32P]Orthophosphate incorporation in lens organ culture indicate that there is little phosphorylation in the inner was carried out in lenses removed from the eye by dissection, regions of the tissue. The protein phosphorylation observed in the outer fiber cells of the tissue gives a distinctly different Abbreviations: IEF, isoelectric focusing; LOCSF, lens outer cortex pattern from that observed in the epithelial cell layer. Most soluble fraction. ofthe observed protein phosphorylation is cAMP dependent. *Preliminary reports were presented at the Seventh International Congress of Eye Research, Alicante, Spain, October 1-7, 1984, and at the Association for Research in Vision and Ophthalmology The publication costs of this article were defrayed in part by page charge Meeting, Sarasota, FL, May 6-10, 1985. payment. This article must therefore be hereby marked "advertisement" tPresent address: Ayerst Laboratories Research, Inc., Princeton, NJ in accordance with 18 U.S.C. §1734 solely to indicate this fact. 08540. 4712 Downloaded by guest on October 2, 2021 Cell Biology: Spector et al. Proc. Natl. Acad. Sci. USA 82 (1985) 4713 leaving a layer of vitreous approximately 4 mm thick on the RESULTS posterior surface. They were immediately placed in 10 ml of a medium containing 1.8 mM CaC12, 5.36 mM KCl, 137 mM The cAMP-dependent phosphorylation ofproteins in LOCSF NaCl, 0.8 mM MgSO4, 5.6 mM glucose, 25 mM Hepes at pH preparations from 1-year-old steer lenses incubated in the 7.4, and [32P]orthophosphate at 0.1 mCi/ml (1 Ci mmol-1), presence of [y-32PJATP has been previously studied by adjusted to 310 milliosmolar with NaCl. After incubation at NaDodSO4/PAGE (3). One of the major cAMP-dependent 370C for 5 hr, the capsule was removed and a soluble fraction phosphorylated products was found with Mr in the 20,000 from the lens outer cortex was prepared as described above range. Working with LOCSF preparations from 4-month-old and analyzed by NaDodSO4/PAGE. calf lenses, under the conditions of this experiment, we Phosphorylated LOCSF preparations were fractionated by observed a similar NaDodSO4/PAGE pattern of cAMP- gel filtration HPLC using a 0.75 x 60 cm TSK G 4000 SW dependent phosphorylated proteins. However, two compo- column (Kratos Analytical Instruments, Ramsey, NJ) oper- nents were resolved in the Mr 20,000 range instead of one ating at a flow rate of 1 ml/min with a buffer containing 20mM (Fig. 1). The Coomassie blue-stained gel profile and its sodium phosphates, 100 mM sodium sulfate, and 50 mM respective radioautograph show the cAMP-dependent 2-mercaptoethanol, pH 6.9. Four milligrams of protein was [32P]phosphate incorporation in two major Coomassie blue- applied in an injection volume of 200 pkl. The elution was stained components with Mr 20,000 and 22,000. monitored with an UV detector at 280 nm. Fractions (0.33 ml) These results suggested that both the A and B chains of were collected and subjected to radioactivity measurement in a-crystallin were being phosphorylated, since this protein is a liquid scintillation counter to obtain the elution profile of the composed of monomers that have apparent molecular radiolabeled material. Fractions to be further analyzed were weights of 22,000 and 20,000 as estimated from mobility on collected, and the protein was precipitated by trichloroacetic NaDodSO4/PAGE (16). Since a-crystallin is present in sig- acid and washed with ethanol and ether as described above. nificant concentration in the lens, has a molecular weight of NaDodSO4/PAGE was carried out according to refs. 8 and approximately 700,000 or more (16), and is much larger than 9, as described previously (3). Mr markers were obtained the other major lens proteins, it can be obtained by HPLC gel from Bio-Rad. Isoelectric focusing (IEF) was carried out filtration procedures (17). Thus, after incubation of calf according to ref. 3. Flat-bed IEF gels containing 2.4% LOCSF preparations with [y-32P]ATP and cAMP, the reac- Ampholine pH 5.0-8.0, 0.6% Ampholine pH 3.5-10.0, and 6 tion mixture was directly fractionated on a TSK G 4000 SW M urea were used.
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