(Received for publication, August 31, 1995; and in revised form, November 20, 1995)
From the
We have used an interaction cloning strategy to isolate cDNAs for sequences that interact with protein kinase C (Chapline, C., Ramsay, K., Klauck, T., and Jaken, S.(1993) J. Biol. Chem. 268, 6858-6861). In this paper, we report a novel sequence, clone 72, isolated according to this method. Clone 72 has a 4.8-kilobase pair open reading frame; antibodies to clone 72 recognize a >200-kDa protein in cell and tissue extracts. Clone 72 message and protein are detected in a variety of tissues. Immunoprecipitation studies demonstrate that clone 72 is the major >200-kDa binding protein described previously in REF52 fibroblasts (Hyatt, S. L., Liao, L., Aderem, A., Nairn, A., and Jaken, S.(1994) Cell Growth & Differ. 5, 495-502). Expression of clone 72 message and protein are decreased in progressively transformed REF52 cells. Since clone 72 is both a protein kinase C (PKC)-binding protein and substrate, decreased levels of clone 72 may influence both the subcellular location of endogenous PKCs as well as signaling events associated with clone 72 phosphorylation. Our results emphasize that the role of PKCs in carcinogenesis may involve several factors, including the quantity and location of the PKCs isozymes and their downstream targets.
Protein kinase C (PKC) ()is a family of
phospholipid-dependent kinases involved in basic cellular functions,
including regulation of growth, differentiation, and gene
expression(1, 2) . The role of individual PKCs in
these processes is not yet known; however, since most cells express
more than one type of PKC, it seems likely that individual PKCs have
unique rather than overlapping functions. All of the PKCs require
phosphatidylserine for maximal activity; however, PKCs can be grouped
according to differences in their dependence on other activators. In
addition to phosphatidylserine, conventional PKCs require calcium and
diacylglycerol, novel PKCs require only diacylglycerol, and atypical
PKCs require nothing more. Several other lipid modifiers of PKC
activity have also been identified, and there is some evidence that
they may selectively influence individual isozyme
activities(1) . Selective isozyme activation in response to
physiological agonists has been noted and may be a result of the
recognized differences in cofactor requirements among the
PKCs(3, 4, 5, 6) .
In addition to isozyme selective activation, isozyme-specific functions may depend on selective substrate recognition. However, only minor differences in substrate specificity among the isozymes have been observed in in vitro assays(7, 8) . Recently, immunofluorescence studies have demonstrated unique subcellular localizations for individual PKCs (9, 10, 11) (data not shown). Thus, targeting of individual PKCs to specific subcellular addresses may be a means of restricting accessibility to substrates and, thereby, provide the mechanism for isozyme-selective phosphorylation events in vivo. To isolate proteins that interact with PKCs with high affinity, we developed an assay for identifying PKC-binding proteins (12, 13, 14) . Subsequently, this assay was adapted to screen expression libraries and isolate cDNA clones for PKC-binding proteins(15) . Binding proteins isolated according to this strategy are also substrates(16) . In this manuscript, we report the full-length sequence of one of these binding proteins, clone 72. The results indicate that clone 72 is widely expressed and is a major PKC-binding protein in REF52 fibroblasts.
Figure 1:
Contig map
of clone 72 cDNA clones and PCR products. The original cDNAs isolated
in the interaction cloning screen (53ORIG and 72ORIG) were overlapping
cDNAs. 5`-RACE of clone 53 produced the 500-bp extension 53EXT1.
5`-RACE of 53EXT1 was used to produce 53EXT2. Concurrently, a rat brain
library was screened with a clone 53 cDNA probe, and brain cDNA (Br
cDNA) was isolated. Additional 3` sequence was obtained by PCR from the
REF52 cDNA library using 3` gene-specific and sequences as primer
pairs. The resulting products (72ext1-5) contained an additional
2.5 kb of coding sequence and 416 bp of 3`-noncoding
sequence.
Figure 2: Primary sequence of clone 72. Consensus sequence of the cDNA clones and PCR products was assembled from the contig map shown in Fig. 1. The translated sequence of the open reading frame (34-4824 bp) is also shown. Position of the peptides in clone 72ORIG and 53ORIG that were phosphorylated by PKC are underlined.
Figure 3: Production of antisera to clone 72. Clone 72ORIG (see Fig. 1) was expressed as a bacterial His-tagged fusion protein and purified by nickel affinity chromatography. The purified protein was used to raise antisera in rabbits. Antisera were immunopurified against the clone 72ORIG expressed sequence. A, Coomassie Blue-stained gel of the clone 72ORIG expressed sequence. B, immunoblots of clone 72ORIG expressed sequence; C, REF52 cell extract probed with affinity-purified antibody raised against clone 72ORIG expressed sequence.
Figure 4: Immunoprecipitation of clone 72 from REF52 cell extracts. Antisera to clone 72ORIG were used to immunoprecipitate proteins from REF52 cell extracts. Immunoprecipitated proteins were blotted to nitrocellulose which was stained with clone 72ORIG antibody (A) or assayed for PKC-binding proteins using the PKC overlay assay (B).
Figure 5:
Tissue distribution of clone 72. A, aliquots of mRNA (2.5 µg) from various tissues were
separated by electrophoresis, blotted, and hybridized to a P-labeled DNA probe of clone 72ORIG. The blot shown was
exposed to film for X days. Positions of molecular weight
markers in kilobase pairs are shown on the right. B,
aliquots of protein (50 µg) from various tissues were separated by
electrophoresis, blotted, and stained with clone 72ORIG antibody.
Positions of molecular weight markers in kilodaltons are shown on the right. Br, brain; H, heart; Ki,
kidney; Li, liver; Lu, lung; Sk, skin; Sp, spleen; Te, testes.
Immunoblots of rat tissues demonstrated a ladder of immunoreactive proteins >200 kDa in most tissues (Fig. 5B). Expression levels were highest in testes and brain. Smaller bands that may be related either to the smaller, related messages or to proteolysis were also detected in most tissues. Thus, clone 72 encodes a high molecular weight PKC-binding protein that is widely expressed in mammalian tissues.
Figure 6:
Transformation-sensitive expression of
clone 72 message in REF52 cells. Aliquots of mRNA (2.5 µg) from
REFA, REFB, REFC, and REFD cells were separated by electrophoresis,
blotted, and hybridized to a P-labeled clone 72ORIG cDNA
probe. The blot shown was exposed to film for days. Positions of
molecular mass markers are shown on the right.
Figure 7: Transformation-sensitive expression of clone 72 protein in REF52 cells. Aliquots of protein (50 µg) from normal and transformed REF cells were separated by electrophoresis and blotted. In A, samples from normal and SV-40-transformed REF52 cells were stained with clone 72ORIG antibody (top), and PKC-binding proteins were detected by the PKC blot overlay assay (bottom). In B, samples from normal and ras-transformed REF cells were stained with clone 72ORIG antibody.
We have used the PKC overlay assay to identify PKC-binding
proteins in cell extracts and to isolate cDNAs for these binding
proteins. Clone 72 is a novel sequence isolated from a REF52 cell
expression library according to its PKC binding properties.
Considerable effort was required to isolate the entire open reading
frame of this large protein. Clone 72 may be identical to a recently
reported sequence, clone 322, which appears to be missing approximately
1000 bp of coding sequence in the 5` end(23) . Other
differences between the reported clone 322 and clone 72 sequences may
be attributed to differences between manual and automated sequencing. ()The translated sequence does not share significant
homology with other proteins in the data bases.
Northern analysis with clone 53 and clone 72 cDNA probes indicate that clone 72 has a broad tissue distribution. These data were substantiated by immunoblot analysis of various tissues with affinity-purified antibody to the clone 72 expressed sequence. Previous studies with the related sequence, clone 322, did not detect a similar tissue distribution profile. The reasons for these discrepancies are not clear at this time. Both studies identified abundant expression of the 6-kb transcript in testes and skin, whereas abundant expression in heart was detected only in this study. The presence of additional messages at 3.0 and 1.2 kb may indicate that alternate forms or closely related sequences are expressed in some tissues.
Several motifs in clone 72 share homology with phosphorylation domains in the PKC substrates neuromodulin and MARCKS(24, 25, 26) . In agreement with this, we found that clone 72 was phosphorylated by PKC in vitro. Although preliminary studies have demonstrated that PKC activation stimulates phosphate incorporation into clone 72 in REF52 cells (data not shown), further studies are required to establish that the phosphorylation is directly due to PKC. Taken together, these data indicate that clone 72 is an abundant PKC substrate and may be important for mediating PKC signals in a variety of cell types.
Immunoblot analysis demonstrated a graded loss of clone 72 protein in SV40- and ras-transformed fibroblasts. These results raise the intriguing possibility that differences not only in the expression of PKCs themselves, but also in the expression of PKC substrates, may contribute to disordered signaling through PKC pathways in transformed cells. Furthermore, since clone 72 can bind PKC with sufficient affinity to be detected in an in vitro overlay assay, it is also feasible that clone 72 functions as a PKC-targeting molecule in vivo. Preliminary immunolocalization studies indicate that clone 72 and certain PKCs both align with fibers in REF52 cells; however, colocalization studies have not yet been done. If clone 72 binds PKC in vivo, loss of clone 72 in transformed cells would also result in loss of PKC targeting. In fact, differential localization of PKCs in normal and transformed cells has been noted (12, 16) . Further studies are required to determine if the differential localization is due to differential expression of PKC-binding proteins in the normal and transformed cells. In principle, inappropriate localization could lead to promiscuous phosphorylation by PKCs and consequently contribute to disordered signaling and loss of growth control.
In summary, our results underscore that understanding the role of PKC signaling in transformation requires analysis not only of the expression of the PKC isozymes and their activation in response to exogenous stimuli, but also analysis of the expression of the downstream targets that mediate the effects of PKC activation on cellular functions, including growth, differentiation, and gene expression.