Protein Kinase A-regulated Instability Site in the 3′-Untranslated Region of Lactate Dehydrogenase-A Subunit mRNA*

Expression of the lactate dehydrogenase A subunit (LDH-A) gene can be controlled by transcriptional as well as posttranscriptional mechanisms. In rat C6 glioma cells, LDH-A mRNA is stabilized by activation and synergistic interaction of protein kinases A and C. In the present study, we aimed to identify the sequence domain which determines and regulates mRNA stability/instability by protein kinase A and focused our attention on the 3′-untranslated region (3′-UTR) of LDH-A mRNA. We have constructed various chimeric globin/lactate dehydrogenase (ldh) genes linked to the c-fos promoter and stably transfected them into rat C6 glioma cells. After their transfection, we determined the half-life of transcribed chimeric globin/ldh mRNAs. The results showed that at least three sequence domains within the LDH-A 3′-UTR consisting of nucleotides 1286–1351, 1453–1471, and 1471–1502 are responsible for the relatively rapid rate of LDH-A mRNA turnover in the cytoplasm. Whereas chimeric globin/ldh mRNAs containing the base sequences 1286–1351 and 1453–1471 were not stabilized by (S p)-cAMPS, an activator of protein kinase A, instability caused by the 1471–1502 domain was significantly reversed. Additional deletion and mutational analyses demonstrated that the 3′-UTR fragment consisting of the 22 bases 1478–1499 is a critical determinant for the (S p)-cAMPS-mediated LDH-A mRNA stabilizing activity. Because of its functional characteristics, we named the 22-base region “cAMP-stabilizing region.”

more energy from the anaerobic pathway by reducing pyruvate to lactate. Investigations into the mechanism of LDH-A gene expression has identified two basic controls consisting of a transcription-regulatory cascade (4,6,11) and a mechanism that regulates the half-life of LDH-A mRNA (4,12), both of which are major determinants of intracellular LDH-A mRNA levels.
Messenger RNA turnover rates fluctuate over a wide range, and it is important to identify and characterize putative stability-regulating mRNA domains and their interacting factors that may be responsible for these functional effects. A great number of reports have demonstrated the existence of such domains and their trans-acting regulatory factors that are critical in determining the half-life of mRNA (13). Several of these studies indicate that the stability of some, but not all, mRNA is determined by specific cis-acting AU-rich domains located in the 3Ј-UTR. For example, a number of mRNAs such as cytokine, lymphokine and protooncogene mRNAs share a common sequence motif with a high content of A and U nucleotides in the 3Ј-UTR (14) and exhibit half-lives in the range of only a fraction of 1 h (15)(16)(17)(18). In addition, attention has focused on modulation of mRNA stability in response to a variety of physiological signals. For instance, histone mRNA stability is regulated by the cell cycle (19) and intracellular iron levels control the stability of transferrin receptor mRNA (20,21). Moreover, manipulation of cells with several different effector agents can alter the steady-state level of mRNA during cell growth or differentiation and may exhibit tissue-specific variations (14). This suggests that in addition to stability-regulating RNA domains effector agent-responsive protein factors may be involved in determining mRNA stability and that they could be subject to regulation by agents that activate second messenger signal pathways.
It has recently been demonstrated that activators of protein kinases A and C are important effectors of mRNA stability regulation in a number of gene systems (14). Although it is known that increased levels of either cAMP or phorbol ester are sufficient signals for increased LDH-A mRNA stability, the molecular mechanisms mediating the effects of cAMP or phorbol ester on mRNA stability have not been defined in detail. Based on our previous data (4,12), we suggested that sequences within the non-coding regions of LDH-A mRNA together with protein kinase-regulated RNA-binding phosphoprotein(s) may play a pivotal role in determining the basal and regulated stability of mRNA (12,22).
In the present study, we have chosen to identify putative cis-regulatory domains within the 3Ј-UTR of LDH-A mRNA that are involved in protein kinases A-mediated mRNA stability regulation. Our initial approach was to express transfected chimeric ␤-globin/ldh 3Ј-UTR constructs and to evaluate the functional effects of protein kinase A on chimeric mRNA stability. Furthermore, applying ribonuclease protection assay to determine the half-life of truncated and mutated fragments of LDH-A 3Ј-UTR, we systematically analyzed the 3Ј-UTR for the presence of (a) sequence domain(s) that cause mRNA instability, and (b) stability-regulatory domain(s) whose activity is modulated by protein kinase A. Our experiments demonstrate that several 3Ј-UTR fragments evoked marked instability of the otherwise relatively stable ␤-globin mRNA. Most importantly, we were able to identify a uridine-rich cAMP-stabilizing region (CSR) responsible for regulating the rate of LDH-A mRNA turnover in response to activators of protein kinase A and the phosphatase inhibitor okadaic acid.

EXPERIMENTAL PROCEDURES
Materials-Nucleic acid-modifying enzymes, acrylamide, nucleoside triphosphates were from Boehringer Mannheim. Radioisotopes were purchased from NEN Life Science Products. Other reagents were of molecular biology grade and purchased from Sigma. Cell culture products were purchased from Life Technologies, Inc. (S p )-Adenosine 3Ј,5Јcyclic monophosphorothioate ((S p )-cAMPS) and (R p )-adenosine 3Ј,5Јcyclic monophosphorothioate ((R p )-cAMPS) were from BIOLOG Life Science Institute.
Synthetic Oligonucleotides-Synthesis and processing of synthetic DNA oligonucleotides and their ligation into the respective plasmid vectors were performed as described previously (23).
Cell Culture-Rat C6 glioma cells (ATCC CCL 107) were maintained as monolayers in Ham's F-10 nutrient medium supplemented with 10% dialyzed fetal calf serum, 50 units/ml penicillin, and 50 mg of streptomycin as described by us (9). All experiments were carried out at about 90% confluence, and serum was withdrawn 16 -18 h prior to addition of various agents.
The rabbit ␤-globin expression vector pRc/FBB was constructed by Dr. D. Chagnovich (Northwestern University) in two steps from plasmids pRc/CMV (Invitrogen) and pBBB (kindly provided by Dr. M. E. Greenberg). To that purpose, pRc/CMV was linearized with BglII and blunt-ended with T4 DNA polymerase. After insertion of a decamer containing a SacII restriction site, the resulting vector was cut with SacII and HindIII to excise the cytomegalovirus promoter and to serve as acceptor for the modified pBBB. For this modification a SacII-Nru I fragment was excised from pBBB and replaced with HindIII linkers. After restriction with SacII, pRc/FBB was created by linking modified pBBB with the modified SacII-HindIII fragment of pRc/CMV. Plasmid pRc/FBB encodes a transcription unit consisting of ␤-globin coding region flanked by the ␤-globin 5Ј-and 3Ј-untranslated regions fused to the c-fos promoter. The sequence and correct orientation of all inserts were verified by restriction and DNA sequence analyses. Sequencing was carried out in both directions by the dideoxynucleotide chain terminator method with specific synthetic oligonucleotides as primers.
Stable Transfection with Expression Vectors and Selection of G418resistant Clones-One day prior to transfection, cell cultures were prepared by seeding 1 ϫ 10 5 cells/60-mm plate in medium containing 10% fetal calf serum. Each plate was treated with 100 g of Lipofectin in 2 ml of Opti-MEM I for 20 min after which 10 g of supercoiled plasmid DNA were added. After 16 h, the Lipofectin solution was replaced with 3 ml of DMEM supplemented with 2% fetal calf serum. Two days following transfection, cells were trypsinized and replated at several dilutions between 1:5 and 1:10 in selective medium containing 0.5 mg/ml G418 (Geneticin). Cells were fed with selective medium every third day until resistant colonies were clearly visible (after about 2 weeks). Individual drug-resistant colonies were subcloned and expanded under selection conditions. RNA Preparation and mRNA Half-life Measurements-Total cytoplasmic RNA was prepared as described (12). Transcripts derived from the glyceraldehyde-3-phosphate dehydrogenase gene and the various transfected chimeric ␤-globin/ldh 3Ј-UTR vectors were detected by RNase protection analysis of 5 g of RNA isolated at various times after serum stimulation and addition of effector agents using 32 P-labeled complementary rabbit ␤-globin antisense probes (24). The complete antisense ␤-globin probe was synthesized with the MAXIscript in vitro transcription kit from the rabbit ␤-globin gene cloned into pBluescript. Radioactivity corresponding to the protected fragments was visualized by autoradiography and quantified using a BAS III FUJI radioanalytic imaging scanner. The half-life of LDH-A mRNA and various chimeric globin/ldh mRNAs was calculated by a nonlinear regression analysis of the results using the InPlot program (GraphPAD Software, San Diego, CA). The decay of chimeric globin/ldh mRNAs from the linker-scanning studies was quantitated using Real-Time PCR detection with a 7700 ABI PRISM Sequence Detection system using the TaqMan PCR reagent kit (Perkin-Elmer) with two fluorogenic probes: 6-carboxyfluorescein as the reporter probe and 6-carboxy-tetramethylrhodamine as the quencher probe (25).
Southern Blot Analysis-Isolation of genomic DNA and Southern blot hybridization analysis were carried out as described (26).

LDH-A 3Ј-UTR Is
Responsible for mRNA Instability-It is known that the presence of AU-rich regions in the 3Ј-UTR may function as destabilizing elements in several mRNAs. Sequence analysis of the LDH-A 3Ј-UTR identifies a 99-nucleotide stretch (nucleotides 1450 -1549), which is relatively AU-rich when compared with the overall nucleotide composition of the 3Ј-UTR. Whether or not this AU-rich region contains a site(s) that determines LDH-A mRNA stabilizing/destabilizing activity and can additionally be modulated through protein kinase signal pathways has so far not been determined.
As a first step to experimentally identify stability/instability elements in the 3Ј-UTR of LDH-A mRNA, we replaced the 3Ј-UTR of ␤-globin mRNA in plasmid pRc/FBB with the entire LDH-A 3Ј-UTR and then determined the rate of decay and half-life of the chimeric globin/ldh mRNA under various experimental conditions. By choosing an expression vector (pRc/ FBB) with a serum-inducible c-fos promoter (15,27,28), we also avoided artifacts that potentially occur when commonly used transcriptional inhibitors such as actinomycin D (29,30) are used to stop ongoing transcription. To perform the decay studies, the pRc/FBB/ldh construct was introduced into rat C6 glioma cells by stable transfection. G418-resistant colonies were pooled to ensure a heterogeneity of integration sites. Quantitative ribonuclease protection assays for each chimeric vector were performed to measure mRNA half-lives. After serum-starving the transfected cells for 25-30 h, pulse induction of the chimeric globin/ldh gene under the control of the c-fos promoter was done by addition of fetal calf serum. Total cytoplasmic RNA was isolated at subsequent time points and ribonuclease protection assays were done. As shown in Fig. 1 (panel A, lane wt ␤-globin), wild-type ␤-globin mRNA was remarkably stable and persisted in the cytoplasm with a half-life of about 21 h (extrapolated from Fig. 1, panel B), similar to data obtained by others (28,(31)(32)(33). In stark contrast, the chimeric ␤-globin/ldh mRNA (panel A, lane Control) decayed at a much faster rate (t 1/2 Ϸ 70 min) (Fig. 1, panel B), similar to the rapid rate of decay of wild-type LDH-A mRNA (t 1/2 Ϸ 55 min) in glioma (4,12). For each chimeric vector the decay data consistently obeyed first-order kinetics. Since the basal mRNA levels may reflect the copy number of integrated globin genes after transfection, we analyzed the copy number of integrated globin genes by Southern blot analysis (not shown). We found that the average number of integrated globin copies is nearly the same for all transfected cells.
By treating the transfected cells with the protein kinase A agonist, (S p )-cAMPS, we found that the activated protein ki-nase had the ability to alter the stability of chimeric globin/ldh mRNA. (S p )-cAMPS significantly prolonged the half-life of hybrid globin/ldh mRNA (panel A, lane Sp-cAMPS) but not that of wild-type globin mRNA (not shown). For instance, the half-life of globin/ldh mRNA increased from about 70 min in untreated cells to about 8 h in (S p )-cAMPS-treated cells (see panel B).
Inasmuch as the above data suggested the involvement of protein kinase A and, hence, protein phosphorylation, we sought additional insight into the significance of potential phosphorylation events by preventing protein phosphorylation through the use of inhibitors of protein kinase A. We used the selective protein kinase A antagonist (R p )-cAMPS to prevent protein kinase activation. The results summarized in Table I show that (R p )-cAMPS blocks protein kinase A-mediated stabilization of globin/ldh mRNA. Furthermore, we used okadaic acid, an inhibitor of protein phosphatases-1 and -2A (34), to shift the overall balance of phospho-/dephosphoproteins in favor of the phosphorylated proteins. Exposure of transfected cells to okadaic acid (20 nM) resulted in a much slower decay and a markedly enhanced stability of globin/ldh mRNA (see Fig. 2).
Thus, the studies show that the half-life of chimeric globin/ ldh mRNA and its regulation are similar, if not identical, to that of wild-type LDH-A mRNA and that the 3Ј-UTR contains all elements needed to (a) destabilize LDH-A mRNA and (b) to convey protein kinase-mediated stability to globin mRNA. We conclude that regulation of LDH-A mRNA stability is an inherent function of its 3Ј-UTR and that it is not, or is only to some minor degree, affected by other regions of the mRNA (such as coding regions and 5Ј-UTR).
LDH 3Ј-UTR Contains at Least Three Determinants of Instability-Based on above data, we proceeded to identify putative regions within the LDH-A 3Ј-UTR that are (a) instrumental in determining instability of LDH-A mRNA and (b) responsible for the protein kinase-mediated stabilization of the mRNA. To that purpose, we generated a series of systematically truncated 3Ј-UTR fragments that were inserted into the unique BglII site located at the junction of the ␤-globin translated and 3Ј-untranslated regions in pRc/FBB (35) (see Fig. 3). This produced vectors with a globin reporter gene, which, upon transcription, yielded unique chimeric globin/ldh mRNAs whose decay characteristics and half-lives were determined. As in each vector the promoter (c-fos) and globin coding regions are identical, the transcripts differ from each other only in their 3Ј-UTR sequence.
In our studies, the density of autoradiographic bands corresponding to various mRNAs was normalized to that of glyceraldehyde-3-phosphate dehydrogenase mRNA, since this mRNA was stable under the experimental conditions regardless of the presence or absence of effector agent. The normalized band of highest density was taken as the starting decay time point. The basal half-lives of the different truncated chimeric globin/ldh mRNAs are shown in Fig. 3. The half-life of several chimeric mRNAs was only slightly lower (between 18 and 20 h) than that of wild-type ␤-globin mRNA (about 21 h), and they decayed less than 10% over the 12-h time course of the experiment (time course not shown). In contrast, insertion of frag-    Table  II. Chimeric mRNAs transcribed from vectors with inserted fragments 1286 -1351 and 1453-1470 were destabilized, but their stability was not affected by treatment of cells with (S p )-cAMPS. In contrast, cells transfected with a vector containing fragment 1453-1527, 1463-1502, or 1463-1527 transcribed a chimeric mRNA that was not only destabilized but responded to (S p )-cAMPS treatment of cells with a significant stabilization of the corresponding globin/ldh mRNAs. When fragment 1463-1502 was further shortened to bases 1471-1502, the resulting chimeric globin/ldh mRNA was destabilized and cells responded to (S p )-cAMPS treatment with stabilization of the mRNA. Further truncation resulted in a loss of (S p )-cAMPS responsiveness (see below). Thus, the data show that fragment 1471-1502 contains the sequences that are necessary for the stabilizing effect of (S p )-cAMPS.
Deletion of Fragment 1478 -1502 Causes Loss of (S p )-cAMP Responsiveness of LDH-A 3Ј-UTR-To expand and confirm the above data, we carried out a stability analysis of LDH-A 3Ј-UTR in which partial base sequences had systematically been deleted from the 3Ј-UTR. Globin 3Ј-UTR in pRc/FBB was removed by BglII/HindIII digestion and replaced with truncated LDH-A 3Ј-UTR fragments. After stable transfection of the vectors into rat C6 glioma cells, the vectors were transcribed, and the decay characteristics of the chimeric globin/ldh mRNAs were analyzed. The half-lives determined by this analysis are summarized in Table III. While deletion of various fragments had little effect on destabilization and (S p )-cAMPS responsiveness, deletion of fragment 1453-1527 and the even shorter fragment 1478 -1502 resulted in increased stability and loss of (S p )-cAMPS responsiveness confirming that a cAMP-responsive instability/stability element is present in fragment 1478 -1502.
Bases 1478 -1499 Are Critical for (S p )-cAMPS-mediated LDH-A mRNA Stabilizing Activity-Having shown that the 1478 -1502 nucleotide sequence was required to achieve (S p )-cAMPS-mediated mRNA stabilization, we further characterized the region by mutational analysis. We inserted bases 1478 -1504 in the correct and reverse orientation into the BglII site of pRc/FBB and analyzed the decay characteristics. Insertion in the correct orientation (1478 -1504wt) caused instability of the chimeric mRNA and stabilization by (S p )-cAMPS (Table  IV), while positioning in the reverse orientation (1478 -1504rev) had no effect on stability or (S p )-cAMPS responsiveness, indicating that the destabilizing and regulatory effects of CSR required a specific polarity of the base sequence.
Several mutational changes throughout the 27-base 1478 -1504 fragment led to a further definition of the active (S p )-cAMPS-responsive base region. Introduction of systematic linker scanning mutations from 1478 through 1502 (mut1 through mut4) and also randomly-placed base mutations (mut5) did not abolish mRNA destabilization (Table IV). However, the (S p )-cAMPS stabilizing effect was completely lost in mut1, -2, -3, and -5. In contrast, the mutations at 1500 -1502 had no effect on (S p )-cAMPS-mediated stabilization. DISCUSSION During recent years, we have provided evidence indicating a transcriptional as well as posttranscriptional regulation of the LDH-A subunit gene. Clues to mechanisms underlying this dual mode of control were provided by our previous identifica-

FIG. 3. Half-life of truncated fragments derived from LDH-A subunit 3-UTR.
Rat C6 glioma cells were stably transfected with pRc/FBB in which the indicated truncated fragments had been inserted into the BglII site located at the junction of the ␤-globin coding and 3Ј-UTR region. After addition of serum, RNA was isolated at various time points up to 16 h. Globin/ldh mRNA decay was assessed by ribonuclease protection assay, and half-lives were determined as described under "Experimental Procedures." Dotted fragments, containing instability element(s) not regulated by (S p )-cAMPS; diagonally and vertically hatched fragments, containing an instability element regulated by (S p )-cAMPS.

TABLE II
Effect of (S p )-cAMPS on the half-life of chimeric globin/ldh mRNAs Rat C6 glioma cells were stably transfected with pRc/FBB in which the listed fragments of LDH-A 3Ј-UTR (with 5Ј and 3Ј BglII ends) had been inserted into the BglII site of pRc/FBB (see Fig. 3). Cells were treated in serum-free medium for 6 h with 0.5 mM (S p )-cAMPS. After addition of serum, RNA was isolated at various time points up to 12 h. Globin/ldh mRNA decay was assessed by ribonuclease protection assay, and half-lives were determined as described under "Experimental Procedures." Results are expressed as mean and S.E. of four separate experiments. tion of cis-acting promoter elements that are instrumental in a protein kinase A-and C-mediated transcriptional regulation (6,7) and by our demonstration of LDH-A mRNA stability regulation through activation of protein kinases A and C signal pathways (12). In order to gain insight into the mechanism of LDH-A mRNA stability regulation, we localized and characterized the 3Ј-UTR sequences responsible for the rapid turnover of LDH-A mRNA and its stabilization via the protein kinase A signal pathway. To this effect, we have transcribed chimeric globin/ldh gene constructs from the c-fos promoter for the purpose of systematically evaluating the contribution of the LDH-A 3Ј-UTR or partial sequences thereof on mRNA stability/ instability as well as the functional effects of protein kinase A. After replacement of the entire ␤-globin 3Ј-UTR with LDH-A 3Ј-UTR, we determined the half-life of the transcribed chimeric globin/ldh mRNA to be 69 Ϯ 4 min. Thus, the half-life of 55 Ϯ 6 min measured previously for wild-type LDH-A mRNA (4,12) compares well with the value measured for chimeric globin/ldh mRNA in the present study. Moreover, activation of protein kinase A by an appropriate agonist exerted similar stabilizing effects on globin/ldh mRNA as reported for wild-type LDH-A mRNA in glioma cells (12). These findings indicate to us that the 3Ј-UTR is the primary determinant of LDH-A mRNA instability/stability and that sequence(s) responsible for determining and regulating LDH-A mRNA stability reside in the 3Ј end of the message.
To localize the sequence domains within the 3Ј-UTR responsible for instability and for stabilization, we have used LDH-A 3Ј-UTR from which small fragments had been systematically deleted as well as truncated and mutated 3Ј-UTR fragments to construct chimeric globin/ldh vectors. After their transfection and transcription, we determined the decay characteristics of the resulting chimeric globin/ldh mRNAs. This experimental approach allowed us to identify two types of destabilizing sequences within the LDH 3Ј-UTR: (i) two regions at base positions 1286 -1351 and 1453-1471, which do not respond to (S p )-cAMPS stimulation; and (ii) an U-rich (53%) 22-base sequence at 1478 -1499, -AUAUUUUCUGUAUUAUAUGUGU-, whose presence in the 3Ј-UTR destabilizes the message and responds to protein kinase A activation with a marked mRNA stabilization. Based on its function, we have named the 22-base sequence "cAMP-stabilizing region" (CSR). The unregulated destabilizing regions (bases 1286 -1351 and 1453-1471) have a uridine content of 24, respectively 40%. Although they markedly reduce the half-life of normally long-lived globin mRNA when inserted as truncated fragments into globin mRNA, they make only a minor destabilizing contribution in globin/ldh mRNA containing the complete LDH-A 3Ј-UTR from which the CSR had been deleted (see for instance Table IV). Thus, the most significant results of the present work consist of identification of a region (bases 1478 -1499) with a twofold function: (a) it acts as an U-rich instability element, and (b) it functions specifically as a dominant stabilizer of LDH-A mRNA half-life in response to activation of the protein kinase A signal transduction pathway.
cis-Acting elements within the 3Ј-UTRs are important points of stability control. One example is the occurrence of a class of short-lived mRNAs that share AU-rich motifs in their 3Ј-UTRs (14). Although a consensus sequence of -AUUUA-was identified as the potential signal for instability (15), it is known that the -AUUUA-sequence or multimers thereof may not be the only signals for destabilization of mRNAs. The CSR is positioned within an AU-rich environment. Although we cannot assess the contribution of the nucleotide environment to the specificity of CSR action at this time, it is conceivable that it may determine a specific secondary structure and RNA conformation necessary for interaction with trans-regulatory proteins.
The present research extents our knowledge of cognate 3Ј-UTR sequences involved in agonist-mediated mRNA stabilization. We know that the turnover rates of a number of mRNA species can be modulated by different biological signals. For instance, treatment of cells with phorbol ester (12, 36 -38), antibodies to cell surface proteins (39), serum (40), steroid and thyroid hormones (41)(42)(43)(44)(45), and cAMP-generating agonists (4, 46 -48) can modulate the half-lives of unstable messages. It is generally accepted that the regulation of mRNA turnover depends on a variety of specific cis-acting sequences and transacting factors (49). The trigger event for mRNA decay consists of several steps involving poly(A) shortening, impaired translation, and nucleolytic cleavage. Additional complexity of the decay mechanisms is added by the nature of the primary and secondary structure of the stabilizing/destabilizing 3Ј-UTR element, the destabilizing nucleolytic enzymes, and by transacting factors that may modulate the interaction of nucleases with the mRNA.
In another paper (22), we have described a complementary experimental approach to the identification of cis-regulatory sequences in the LDH-A 3Ј-UTR by searching for putative trans-regulatory components, e.g. proteins that bind to the CSR and may be involved in regulating LDH-A mRNA stability. We were able to identify four CSR-binding proteins whose binding

TABLE IV
Effect of (S p )-cAMPS on the half-life of wild-type and mutated chimeric globin/ldh mRNAs Rat C6 glioma cells were stably transfected with pRc/FBB in which the listed 3Ј-UTR fragments had been inserted into the BglII site of pRC/FBB (see Fig. 3). Stimulation with (S p )-cAMPS and mRNA decay analysis were done as described in legend of activity was up-regulated after cAMP-mediated activation of protein kinase A, implying a correlation between phosphorylative modification of the proteins and their putative mRNA-stabilizing activity. While we speculate that mRNA stability may be achieved through interaction of RNA domains with phosphorylated RNA-binding proteins, we must consider that the degradative process may be controlled through phosphorylative and functional modification of nucleolytic enzymes. However, this scenario makes several assumptions: (a) the nucleolytic enzyme(s) is LDH mRNA sequence-specific, (b) the nuclease(s) is phosphorylated by protein kinase A leading to its deactivation, (c) the nuclease(s) may be specifically recognized by a component(s) of the activated signal transduction systems and as a result of the interaction be functionally modified.
Although we believe this scenario to be unlikely, it should be noted that Bandyopadhyay and co-workers (50) have observed that polysome preparations may contain a nuclease activity that is subject to inhibition by a soluble cytoplasmic factor. Thus, the possibility that the activity and specificity of nucleolytic enzymes may be regulated by protein kinase-modified factors needs further study.