Purification and Characterization of a Novel Physiological Substrate for Calcineurin in Mammalian Cells*

Although the calcium/calmodulin-regulated protein phosphatase calcineurin has been shown to play a role in a number of intracellular processes, relatively few of the downstream phosphoproteins that are dephosphorylated by this enzyme in cells have been described. Calcineurin was previously shown to play a role in amylase secretion by rat pancreatic acinar cells and to specifically dephosphorylate a 24-kDa cytosolic protein. The present study describes the purification and characterization of this novel phosphoprotein, termed CRHSP-24 (calcium-regulatedheat-stable protein with a molecular mass of 24 kDa). Microgram quantities of CRHSP-24 were purified from a large-scale rat pancreas preparation in a procedure involving heat and acid precipitation, anion-exchange chromatography, preparative electrophoresis, electroelution, and two-dimensional electrophoresis. Internal amino acid sequence was obtained from two peptides following trypsin digestion and high pressure liquid chromatography. Both sequences matched with 100% identity nucleotide sequences of expressed sequence tags from human placenta and rat PC-12 cells. Two CRHSP-24 transcripts of 0.7 and 2.9 kilobases were detected in multiple rat tissues by Northern analysis, whereas a single 24-kDa protein was observed by Western blotting. The CRHSP-24 protein is 147 amino acids in length, is composed of nearly 14% proline, and is phosphorylated entirely on serine residues. Western analysis and 32P metabolic labeling of acini revealed CRHSP-24 to be maximally phosphorylated in control cells and to undergo a rapid sustained dephosphorylation on at least 3 serine residues in response to calcium-mobilizing stimuli. Dephosphorylation of CRHSP-24 was completely inhibited by pretreatment of acini with cyclosporin A or FK506. Furthermore, the inhibitory effects of FK506 were blocked by excess rapamycin. The ubiquitous expression of CRHSP-24 in rat tissues suggests that this novel calcineurin substrate plays a common role in calcium-mediated signal transduction.

Calcineurin, also known as protein phosphatase-2B, is unique among the serine/threonine protein phosphatases because its activity is highly regulated by calcium and calmodulin. This enzyme is a heterodimer composed of a 19-kDa regulatory subunit and 60-kDa catalytic subunit, the latter of which binds calmodulin (reviewed in Refs. [1][2][3][4][5][6]. Both the regulatory subunit and calmodulin are calcium-binding proteins, each containing EF-hand motifs. As the holoenzyme is activated by high nanomolar/low micromolar concentrations of calcium in vitro, calcineurin is believed to be relatively inactive in cells under basal conditions of low intracellular calcium and then becomes activated following stimulation with calcium-mobilizing agonists (7).
A pivotal role for calcineurin in regulating immune cell function has emerged based on the discovery that the immunosuppressants cyclosporin A (CsA) 1 and FK506 are specific inhibitors of calcineurin in T cells (reviewed in Refs. 4 -6 and 8). CsA and FK506 form complexes with their respective intracellular binding proteins, cyclophilin A and FKBP12, which then bind to and inhibit calcineurin phosphatase activity (9 -11). Inhibition of calcineurin in T cells blocks the dephosphorylation of the transcription factor NFAT, thereby inhibiting calcium-stimulated gene transcription (12). Use of CsA and FK506 has also implicated calcineurin in a number of other cellular processes, including secretion (13)(14)(15)(16); endocytosis, ion channel modulation, cytoskeletal organization, neurite outgrowth, and transcription (reviewed in Ref. 16); mRNA stabilization (17); Na ϩ / K ϩ -ATPase activity (18); and apoptosis (19,20).
Despite the numerous studies addressing the function of calcineurin in cells, little is known of the specific biochemical events associated with its activation. The most well characterized calcineurin substrates are the protein phosphatase-1 inhibitors DARPP-32 (18,19) and inhibitor-1, which are dephosphorylated on a specific threonine residue (3)(4)(5)(6), and NFAT, which is dephosphorylated on multiple serine residues (12). Other less well characterized phosphoproteins reported to be dephosphorylated by calcineurin include the inositol trisphosphate receptor (22), the microtubule-associated protein MAP2 (23), the synaptic nerve terminal protein dynamin (24), the dual leucine zipper-bearing kinase (25), and the transcription factor ELK-1 (26). Thus, in contrast to the apparent multitude of cellular substrates for the type 1 and 2A serine/threonine phosphatases in cells (3)(4)(5)(6), relatively few in vivo cellular targets of calcineurin have been described, especially outside of the nervous system.
Pancreatic acinar cells are among the most well character-ized electrically non-excitable cell types that utilize calcium as a primary mode of intracellular signaling. These highly differentiated epithelial cells are responsible for the synthesis, packaging, storage, and regulated secretion of digestive enzymes (27). CsA was previously shown to inhibit calcineurin activity and calcium-stimulated secretion in acutely isolated rat acinar cells (15). Using 32 P metabolic labeling and two-dimensional electrophoresis, it was found that calcineurin inhibition specifically blocked the secretagogue-induced dephosphorylation of a 24-kDa cytosolic phosphoprotein while having no discernible effects on other phosphoproteins. This protein was further characterized as a calcineurin substrate by demonstrating its selective in vitro dephosphorylation in acinar cell lysates prepared in the presence of okadaic acid and EDTA to inhibit other serine/threonine protein phosphatases (15). The present study describes the purification and characterization of this novel phosphoprotein, termed CRHSP-24 for calcium-regulated heatstable protein with a molecular mass of 24 kDa.

EXPERIMENTAL PROCEDURES
Materials-Poly(A) RNA from rat pancreas and the rat multi-tissue poly(A) RNA Northern blot were purchased from CLONTECH (Palo Alto, CA). Cyclosporin A was a gift from Sandoz Pharmaceuticals (East Hanover, NJ). FK506 was purchased from BIOMOL Research Labs Inc. (Plymouth Meeting, PA), and rapamycin from LC Laboratories (Worburn, MA). The pGEX-Kt expression vector was a gift from Dr. J. Dixon. Human expressed sequence tag clone 143057 containing CRHSP-24 was provided by the WashU-Merck EST Project (St. Louis, MO) and distributed through the Integrated Molecular Analysis of Genome Expression Consortium, Lawrence Livermore National Laboratory. Rat expressed sequence tag clone 106032 was provided by the Institute for Genomic Research (Gaithersburg, MD). Antibody production was conducted at the Pocono Rabbit Farm and Laboratory Inc. (Canadensis, PA).
Isolation of Rat Acini-Pancreatic acinar cells were isolated from Sprague-Dawley rats (28) and labeled with [ 32 P]orthophosphate as described previously (15,29,30). CsA and rapamycin were dissolved in ethanol, and FK506 in dimethyl sulfoxide. These compounds were added to cells, creating a final vehicle concentration of 0.1%; control cells received equal final concentrations of vehicle.
Phosphoamino Acid Analysis-Heat-stable protein prepared from 32 P-labeled acinar cells was separated by two-dimensional PAGE (15,29,30), and the phosphoamino acid content of the CRHSP-24 isoforms was analyzed as described previously (30).
Purification of CRHSP-24 -Purification was conducted in batches of 100 or 200 rat pancreases. Each pancreas was removed, immediately frozen in liquid nitrogen, and stored for 24 h at Ϫ40°C before use. Frozen pancreases were homogenized in 5 volumes of lysis buffer containing 20 mM Tris, pH 7.8, 0.25 M sucrose, 2 mM EDTA, 50 mM NaF, 2 mM sodium pyrophosphate, 15 mM 2-mercaptoethanol, 1 mM benzamidine, 0.5 mM phenylmethylsulfonyl fluoride, 5 g/ml leupeptin, 5 g/ml pepstatin, and 0.1 mg/ml soybean trypsin inhibitor and centrifuged at 150,000 ϫ g for 1 h. Heat-stable protein was prepared from the soluble fraction as described (yielding ϳ125 mg of heat-stable protein/100 pancreases) (30). Heat-stable protein isolated from 32 P-labeled acinar cells was then added to the unlabeled material in order to identify CRHSP-24 throughout the purification procedure. The combined extracts were adjusted with 100% trichloroacetic acid to a final concentration of 0.25% (w/v) for 1 h at 4°C. Precipitated material was removed by centrifugation at 3000 ϫ g for 20 min. The soluble fraction was further adjusted to 2.5% trichloroacetic acid for 1 h, and the precipitated protein (ϳ20 mg of total protein/100 pancreases), containing CRHSP-24, was recovered by a second centrifugation. The pellet was washed with 100% ethanol and then suspended in 10 -20 ml of 20 mM Tris buffer, pH 7.8, creating a final pH of 6.8 at room temperature. Using an Amersham Pharmacia Biotech fast protein liquid chromatography apparatus, the extract was loaded on to a Mono-Q anion-exchange column that had been equilibrated in 20 mM Tris, pH 7.8, and then eluted with a 0 -200 mM NaCl linear gradient at a flow rate of 0.5 ml/min. CRHSP-24 was recovered in the 0 -160 mM NaCl fraction (containing ϳ2 mg of total protein/100 pancreases). Eluted proteins were filtered through a Centriplus M r 100,000 cutoff membrane and then concentrated in a Centriprep-10 concentrating tube. Proteins were separated on multiple lanes (350 -400 g/lane) of 12.5% SDS gels using a Bio-Rad II preparative electrophoresis apparatus. Following SDS-PAGE, the gels were dried and autoradiographed. The portion of each gel corresponding to CRHSP-24 was excised with a razor and rehydrated in electroelution buffer (25 mM Tris base, 192 mM glycine, and 0.1% SDS). Proteins were eluted from the gel pieces using a Bio-Rad electroelution apparatus and then electrodialyzed overnight in the absence of SDS. Approximately 12 g of bovine serum albumin was added to the combined eluants, and the proteins were concentrated in a Centricon-10 tube. SDS was further removed by passing the sample through an Extractigel column (Pierce). The sample was then reconcentrated and diluted in two-dimensional PAGE solubilization buffer (29). Using this procedure, proteins that had been purified from a total of 600 rat pancreases were combined, run on two separate two-dimensional PAGE gels, and transferred to polyvinylidene difluoride membranes. The membranes were stained with 0.1% Coomassie Brilliant Blue in 40% methanol and 1% acetic acid and then destained in 50% methanol and washed extensively with water. CRHSP-24 isoforms were excised and digested with trypsin (31). The resulting peptides were separated by reversed-phase HPLC as described (30). Amino acid sequencing was conducted at the Michigan State University Protein Sequencing Facility using an Applied Biosystems Model 494 automated protein sequencer.
CRHSP-24 Fusion Protein-The 441-nucleotide coding sequence of rat CRHSP-24 was inserted into a glutathione S-transferase (GST) pGEX-Kt bacterial expression vector as described previously (30). Nucleotide sequencing verified that the coding sequences for GST and CRHSP-24 were in frame. GST-CRHSP-24 fusion protein was produced in Escherichia coli BL21/DE3 by induction with 0.2 mM isopropyl-␤-Dthiogalactopyranoside for 16 h at room temperature. Recombinant CRHSP-24 was purified from bacterial lysates by glutathione affinity chromatography and thrombin cleavage as described previously (30).
Western and Northern Blotting-Western analysis was conducted as described previously (30). Isoelectric focusing of whole cell lysates prepared in two-dimensional sample buffer was conducted using a Bio-Rad mini-isoelectric focusing cell as recommended by the manufacturer. Anti-rat polyclonal GST-CRHSP-24 antiserum was characterized and used at a final dilution of 1:1000 in combination with a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:5000). Northern analysis was conducted using the 32 P-labeled rat CRHSP-24 cDNA as a probe as described (32).
Immunofluorescence Methods-Rat pancreatic acini were fixed on ice for 2 h in phosphate-buffered saline (PBS) containing 4% formaldehyde, prepared from paraformaldehyde. Subsequently, acini were rinsed in PBS, cryoprotected in sucrose, and frozen after centrifugation in a mixture of 20% sucrose and Tissue-Tek OCT embedding medium (Miles Laboratories Inc., Elkhart, IN) as described previously (33). Procedures for immunofluorescence microscopy with 5-m-thick cryostat sections were as described previously in detail (33). Primary and secondary antibodies were diluted in PBS containing 2% normal goat serum and 0.2% Triton X-100. Affinity-purified rabbit polyclonal antibodies to CRHSP-24 were used at dilutions ranging from 0.5 to 1 g/ml. After antibody incubations and rinsing, sections were mounted under coverslips with a 3:1 mixture of glycerol and PBS containing 4 mg/ml pphenylenediamine and viewed by conventional epifluorescence microscopy (Leitz Aristoplan). Images were digitized and processed using Photoshop 3.0 software (Adobe, Mountain View, CA).

RESULTS AND DISCUSSION
CRHSP-24 Is Phosphorylated on Serine Residues-Based on its isoelectric focusing pattern, CRHSP-24 was determined to be phosphorylated on multiple residues in control cells and to undergo a pronounced alkaline shift in response to the calciummobilizing secretagogue cholecystokinin (CCK) (Fig. 1) or treatment with calcium ionophore (data not shown). In the basal state, CRHSP-24 was maximally phosphorylated and predominantly present as its most acidic isoform (Fig. 1, spot 1, pI 6.2). Following stimulation of cells with CCK, there was a large decrease in the phosphate content of spot 1, with significant increases in the intensities of three more alkaline isoforms of CRHSP-24 (spots 2-4, pI 6.3, 6.5, and 6.8, respectively). Phosphoamino acid analysis of each of the CRHSP-24 isoforms before and after CCK treatment revealed the protein to be phosphorylated entirely on serine residues (Fig. 1). The absence of tyrosine phosphorylation was confirmed by immunoblotting with anti-phosphotyrosine antibodies (data not shown). Collectively, the presence of the four phospho isoforms of CRHSP-24 as seen by isoelectric focusing indicates that the protein is phosphorylated on a minimum of 4 separate serine residues in acinar cells.
Purification and Identification of CRHSP-24 -Efforts to identify CRHSP-24 using specific antibodies to candidate phosphoproteins were unsuccessful, suggesting that CRHSP-24 was novel. Therefore, the amino acid sequence of CRHSP-24 was elucidated by purifying the protein from a large-scale preparation of over 600 rat pancreases. Purification was facilitated by the thermal stability of CRHSP-24 as well as its high solubility in aqueous solutions. As a final step in the procedure, CRHSP-24 was immobilized to polyvinylidene difluoride membrane and detected by staining with Coomassie Brilliant Blue dye and autoradiography (data not shown). Microgram quantities of CRHSP-24 were obtained from each of the three most acidic phospho isoforms of the protein (Fig. 1, spots 1-3). These pieces of membrane were combined and treated with trypsin; the resulting peptides were separated by reversed-phase HPLC ( Fig. 2A). Eluant fractions containing CRHSP-24 peptides were submitted for microsequence analysis. High quality sequence was obtained from two peptides of 19 and 20 amino acids in length.
A search of the GenBank TM Data Bank revealed that both of the purified CRHSP-24 amino acid sequences were positioned in the same reading frame of an expressed sequence tag from a human placenta cDNA library. Upon sequencing, this 722-base pair cDNA was found to contain a Kozak consensus sequence (34) positioned 12 nucleotides from the 5Ј-end, which aligns it with a TAG stop codon beginning at nucleotide 552. This de-fined coding sequence predicts a 147-amino acid protein containing both of the purified rat CRHSP-24 peptides positioned contiguously within the molecule. An expressed sequence tag from rat PC-12 cells showing high homology to the human clone was subsequently identified. This 2.9-kilobase cDNA was found to code for a protein having Ͼ96% homology to the human protein and, likewise, contained both of the purified peptide sequences (Fig. 2C). In addition to an expanded 3Ј-untranslated region, the larger rat cDNA has a 30-nucleotide 5Ј-untranslated region that contains no other translational start sites.
In the nonphosphorylated state, rat CRHSP-24 has a calculated molecular mass of 15.8 kDa and an isoelectric point of 7.8. CRHSP-24 is predicted to be abundantly hydrophilic, consistent with its presence in the soluble fraction following high speed centrifugation. It is rich in proline, containing 20 residues that account for nearly 14% of the protein by frequency. This high proline content is especially noted in the amino terminus, where a stretch of 7 prolines is parted by a single glutamine residue. Finally, rat CRHSP-24 also contains 14 serine, 13 glycine, and 13 valine residues, which collectively constitute Ͼ27% of the protein.
The CRHSP-24 protein has an ϳ62% identity to the predicted coding region of a recently identified cDNA that encodes a putative double-stranded RNA-binding protein termed PIP-PIN (35). Similar to CRHSP-24, PIPPIN is a small (154 amino acids) hydrophilic protein that, when expressed in vitro, undergoes anomalous migration on SDS-PAGE. The highest identity between the two proteins is located carboxyl-terminal to the first 40 amino acids of CRHSP-24; conversely, a much lower identity is noted in the amino termini of the two molecules. Unlike the broad tissue distribution of CRHSP-24 (see below), the PIPPIN mRNA was found exclusively in rat brain. In addition, PIPPIN is predicted to contain two double-stranded RNA-binding domains that are partially conserved in CRHSP-24. Within one of these domains, CRHSP-24 contains a 19amino acid region (residues 73-92) with 73% identity to the E. coli cold-shock proteins. Bacterial cold-shock proteins are a family of low molecular mass (ϳ70 amino acids) DNA-and RNA-binding proteins that are believed to function as transcriptional and/or translational regulatory proteins (36).
Tissue Distribution of CRHSP-24 in Rat-The rat CRHSP-24 cDNA was used to probe a Northern blot of rat pancreas poly(A) mRNA (Fig. 3A). Two CRHSP-24 transcripts of ϳ2.9 and 0.7 kilobases were identified. The larger transcript was similar in size to the rat cDNA, suggesting that this clone potentially represents a full-length CRHSP-24 mRNA. Similarly, the smaller 0.7-kilobase message was consistent with the size of the human cDNA. An analysis of mRNA from various rat tissues indicated that both of the CRHSP-24 transcripts were present in all organs tested. CRHSP-24 mRNA was particularly abundant in pancreas, testis, liver, and lung, whereas lower levels were found in spleen, brain, and heart. Much lower amounts were seen in kidney and skeletal muscle. Similar results were obtained when analyzing the relative expression of the CRHSP-24 protein (Fig. 3B). Immunoblotting using specific anti-rat CRHSP-24 antiserum (see below) indicated a ubiquitous expression of a single 24-kDa protein in rat. The presence of a single protein in pancreas and other tissues using a polyclonal antiserum raised against the full-length CRHSP-24 protein suggests that both of the mRNA transcripts detected by Northern blotting potentially code for the same protein and may differ in their untranslated regions. Alternatively, it is also possible that the smaller CRHSP-24 transcript codes for a homologous or alternatively spliced protein that is not recognized by the antibodies. The highest levels of FIG. 1. CRHSP-24 is phosphorylated on multiple serine residues. Heat-stable proteins from control and cholecystokinin-treated (1 nM, 5 min) 32 P-labeled pancreatic acinar cells were separated by twodimensional electrophoresis and transferred to polyvinylidene difluoride membrane. Upper panels, phosphorylated CRHSP-24 was detected by autoradiography. Lower panel, CRHSP-24 phospho isoforms were excised from the membrane and subjected to phosphoamino acid analysis. Positions of phosphoserine, phosphothreonine, and phosphotyrosine standards are indicated on the right. CRHSP-24 protein expression occurred in pancreas, parotid gland, liver, testis, and lung, whereas the lowest levels were found in stomach, small intestinal mucosa, and kidney.
Bacterial Expression of CRHSP-24 and Production of Specific Antiserum-The predicted coding sequence from the rat CRHSP-24 cDNA was subcloned into a pGEX-Kt expression vector and expressed in bacteria as a GST fusion protein.
Polyclonal rabbit antisera raised against recombinant GST-CRHSP-24 recognized a 24-kDa protein in acinar cell lysates (Fig. 4A, lane 2), which was predominantly associated with the soluble fraction prepared from a 100,000 ϫ g centrifugation (compare lanes 3 and 4) and highly enriched in a heat-stable extract from acini (lane 5). This subcellular localization was unchanged by CCK treatment of acini (data not shown). Specificity of the antiserum was demonstrated by competitively inhibiting the 24-kDa signal with the addition of an excess of recombinant CRHSP-24 protein to the buffer prior to immunoacid shown below each. Positions of the purified rat peptides are underlined. C, comparison of human and rat CRHSP-24 amino acid sequences. The asterisks denote identical amino acids. blotting (lane 6). Using a standard curve generated by immunoblotting various amounts of recombinant rat CRHSP-24 (data not shown) and comparing it with the level of expression in a cell lysate, it was estimated that CRHSP-24 represents ϳ0.01% of total acinar cell protein.
Immunoblotting was also conducted following two-dimensional electrophoresis of 32 P-labeled proteins from control and CCK-stimulated acini (Fig. 4B). Overlaying the autoradiograph of 32 P-labeled CRHSP-24 with the film from the immunoblot revealed that the antiserum recognized all of the phosphorylated isoforms of CRHSP-24. Furthermore, the pattern of the CCK-induced alkaline shift in CRHSP-24 seen by 32 P labeling was readily apparent by immunoblotting, indicating that the affinity of the antiserum for CRHSP-24 was not altered by the phosphorylation state of the protein.
Similar to the subcellular distribution of the protein detected by Western blotting, immunohistochemical localization of CRHSP-24 using affinity-purified antibodies showed a diffuse pattern of cytosolic staining that was most intense in the basal cytoplasm (Fig. 5). Little or no staining was observed in nuclei and secretory granules (Fig. 5). Specificity of CRHSP-24 staining was evidenced by the total loss of fluorescence seen following preincubation of the antibody with a molar excess of antigen (data not shown). A similar diffuse cytosolic localization of CRHSP-24 was also detected in nerve growth factor-differentiated PC-12 cells (data not shown). The overall pattern of CRHSP-24 localization was unchanged by a 30-min KCl depolarization of PC-12 cells or CCK treatment of acini (data not shown).
Immunosuppressants Define a Role for Calcineurin in CRHSP-24 Dephosphorylation-As CRHSP-24 was initially identified as a calcineurin substrate in acinar cells (15), the effects of the immunosuppressants CsA, FK506, and rapamycin to influence its phosphorylation were examined by immunoblotting following isoelectric focusing (Fig. 6). Treatment of acinar cells with CsA (1 M) or FK506 (10 nM) either alone or in combination with rapamycin (2 M) had no effects on CRHSP-24 phosphorylation. In contrast, treatment with 1 nM CCK or the calcium ionophore ionomycin (data not shown) stimulated a marked shift in CRHSP-24 to its three more alkaline isoforms, indicative of its dephosphorylation. The CCK-induced dephosphorylation was seen as early as 30 s and was sustained for up to 30 min in the presence of agonist (data not shown). Pretreatment of cells with CsA or FK506 prior to CCK fully inhibited the dephosphorylation of CRHSP-24, whereas rapamycin pretreatment had no effects. Conversely, excess rapamycin completely reversed the effect of FK506 to inhibit the dephosphorylation of CRHSP-24, but was unable to block the inhibitory effects of CsA. These results are in accordance with previous studies demonstrating that FK506 and rapamycin are ligands for the same intracellular receptor, yet act on different enzymes within cells, namely calcineurin and RAFT1, respectively (40).
Previously, a resistance of acinar cells to calcineurin inhibition by FK506 was reported (15). This resistance was speculated to be due to a reduced concentration of FKBP12 in acini. Subsequently, it was shown that rapamycin, which also acts through FKBP12, is a potent and specific inhibitor of p70 S6 kinase in acini (41). Furthermore, p70 S6 kinase inhibition by rapamycin was reversed by pretreatment with excess FK506, demonstrating that these compounds are ligands for the same intracellular binding protein, but act on different signaling pathways in acini. Whether the FK506 resistance was due to a different source of the compound, its nonspecific binding to buffer components or lab ware, its hydrophobic aggregation in aqueous solution when used at micromolar concentrations, or a reversal of the FK506-FKBP12-calcineurin complex upon preparation of the lysates is uncertain. The present results are consistent with the effects of rapamycin and FK506 on p70 S6 kinase activity in acini and, furthermore, are in close agreement with the pharmacological characteristics of these compounds in immune cells. Collectively, these data strongly support a role for calcineurin in regulating CRHSP-24 phosphorylation in acini.
CRHSP-24 Phosphorylation-The alkaline shift of CRHSP-24 seen following a stimulated increase in intracellular calcium is completely inhibited by pretreatment with CsA or FK506 and is perfectly reproduced in vitro by calcineurin treatment (15), suggesting that at least 3 of its serine residues are susceptible to dephosphorylation by the enzyme. The absence of any additional more alkaline forms of native CRHSP-24 on immunoblot analysis also suggests that 1 of the phosphoserines is not dephosphorylated by calcineurin. Six of the 14 serine residues of CRHSP-24 are located within the first 60 amino acids, with the sequence surrounding serines 30 and 32 forming a consensus site for potential phosphorylation by a number of protein kinases, including the proline-directed kinases. Currently, the specific serine residues that are phosphorylated on CRHSP-24 as well as the specific CRHSP-24 kinase(s) are being investigated.
The regulated phosphorylation of CRHSP-24 is similar to that of NFAT in T cells. Both proteins are maximally phosphorylated on serine residues in resting cells and rapidly dephosphorylated by calcineurin following a rise in free cellular calcium. Like NFAT, CRHSP-24 appears to be phosphorylated by a constitutively active kinase in acini. This is supported by metabolic labeling, showing that under basal conditions, CRHSP-24 will incorporate large amounts of 32 P during a 1-h incubation of cells with radiolabeled orthophosphate. Furthermore, 32 P labeling in the presence of CsA has little effect on the basal phosphorylation of CRHSP-24 (data not shown), indicating that it is also constitutively dephosphorylated by a serine phosphatase other than calcineurin. These results denote a high basal turnover rate of phosphate on CRHSP-24 and also underscore that its phosphorylation is a highly regulated process in acini.
Concluding Remarks-Based on amino acid homologies, CRHSP-24 may play a role as a transcriptional or translational regulatory protein in acinar cells. However, in preliminary experiments, no nucleotide binding activity of either the native or recombinant CRHSP-24 protein has been detected. It should also be noted that nucleotide binding of the cloned PIPPIN protein was not demonstrated in its initial characterization, but rather was reported as a putative property of the molecule (35). In addition, the presence of CRHSP-24 in the soluble fraction of acinar cell lysates seen by immunoblotting, together with its diffuse immunolocalization within cells and absence from the nuclei of acini and PC-12 cells, argues against a transcriptional role for the protein. Alternatively, CsA-mediated calcineurin inhibition in acinar cells was associated with a significant decrease in calcium-stimulated amylase secretion (15). Moreover, both CCK-stimulated secretion and CRHSP-24 dephosphorylation were inhibited over a similar concentration range of CsA (15). As CCK is a major physiological regulatory hormone of exocrine pancreas with effects on all aspects of acinar cell metabolism, including secretion, transcription, and translation, a role for CRHSP-24 in one of these processes is highly likely. Clearly, the elucidation of the functional significance of CRHSP-24 in acini will provide valuable insight into the physiological and biochemical mechanisms of Ca 2ϩ /calcineurin-mediated signal transduction in mammalian cells.
FIG. 6. Immunosuppressants define a role for calcineurin in regulating CRHSP-24 phosphorylation. Acini were treated with immunosuppressants for 15 min prior to the addition of cholecystokinin (1 nM, 2 min). Rapamycin (Rapa.) was added 5 min prior to the addition of CsA or FK506. Whole cell lysates (30 g/lane) were separated by isoelectric focusing and transferred to nitrocellulose membrane. CRHSP-24 phosphorylation states were analyzed by immunoblotting using anti-rat GST-CRHSP-24 serum (1:1000), which resolves the four forms indicated on the two-dimensional gels in Figs. 1 and 4B. Note that the alkaline shift to isoforms 3 and 4 induced by CCK is blocked by CsA and FK506 and that the FK506 effect is reversed by rapamycin.