(Received for publication, May 31, 1995; and in revised form, July 20, 1995)
From the
We recently cloned a partial cDNA (35H) for a protein kinase C
(PKC) binding protein from a rat kidney cDNA library and demonstrated
that it is a PKC substrate in vitro (Chapline, C., Ramsay, K.,
Klauck, T., and Jaken, S.(1993) J. Biol. Chem. 268,
6858-6861). Additional library screening and 5` rapid
amplification of cDNA ends were used to obtain the complete open
reading frame. Amino acid sequence analysis, DNA sequence analysis, and
Northern analysis indicate that 35H is a unique cDNA related to -
and
-adducins. Antisera prepared to the 35H bacterial fusion
protein recognized two polypeptides of 80 and 90 kDa on immunoblots of
kidney homogenates and cultured renal proximal tubule epithelial cell
extracts. The 35H-related proteins were similar to
- and
-adducins in that they were preferentially recovered in the Triton
X-100-insoluble (cytoskeletal, CSK) fraction of cell extracts and were
predominantly localized to cell borders. Phorbol esters stimulated
phosphorylation of CSK 35H proteins, thus emphasizing that sequences
isolated according to PKC binding activity in vitro are also
PKC substrates in vivo. The phosphorylated forms of the 35H
proteins were preferentially recovered in the soluble fraction, thus
demonstrating that phosphorylation regulates their CSK association and,
thereby, their function in regulating cytoskeletal assemblies. We have
isolated another PKC binding protein partial cDNA (clone 45) from a rat
fibroblast library with substantial homology to
-adducin. Antisera
raised against this expressed sequence recognized a protein of 120 kDa,
the reported size of
-adducin, on immunoblots of renal proximal
tubule epithelial cell extracts. A 120-kDa protein that cross-reacts
with the clone 45 (
-adducin) antisera coprecipitated with 35H
immunecomplexes, indicating that
-adducin associates with 35H
proteins in vivo. Taken together, these results indicate that
35H is a new, widely expressed form of adducin capable of forming
heterodimers with
-adducin. We propose naming this adducin
homologue
-adducin.
Protein kinase Cs are a heterogeneous group of
phospholipid-dependent kinases important for cell growth and
differentiated functions (reviewed in (1) ). The family can be
divided into three categories based on enzymatic properties. The
conventional or Group A PKCs ()are calcium-dependent kinases
whose activities are stimulated by diacylglycerol or phorbol esters.
The novel or Group B PKCs are calcium-independent but still
diacylglycerol-stimulatable. The atypical or Group C PKCs are calcium-
and diacylglycerol-independent. Most cells express more than one type
of PKC, which implies that PKCs have unique rather than overlapping
functions.
Activation of Group A and B PKCs is regulated by receptor-mediated production of diacylglycerol through phospholipase C or D pathways(1) . In many cases, activation correlates with PKC redistribution from soluble to particulate fractions. However, in other cases, evidence for activation in the absence of measurable translocation has been noted (2, 3, 4, 5) . The correlation between translocation and activation is further confused by the fact that PKC inhibitors such as staurosporine can also cause PKC redistribution(6, 7) . Thus, redistribution does not necessarily reflect PKC activation under all conditions.
An alternative method for studying PKC activation is to monitor the phosphorylation state of PKC substrates. For example, phosphorylation of MARCKS, a major PKC substrate in many cells, has been useful for demonstrating PKC activation in response to a variety of physiological agonists(8) . Our goal has been to identify a panel of high affinity substrates for PKCs that can be used as reporter systems to monitor PKC activity in response to external stimuli and in pathological processes. To this end, we developed a blot overlay assay to identify PKC binding proteins. In subsequent studies, we demonstrated that MARCKS and other PKC substrates interact with PKCs in this in vitro assay (9, 10) and suggested that, in general, binding proteins are also substrates. The overlay assay is based on a phospholipid-dependent, high affinity interaction between PKCs and recognition sites on other proteins (substrates)(11) . With slight modifications, the assay can also be used in an interaction cloning strategy to isolate expressed cDNAs for PKC substrates (12) .
In our original report on interaction cloning of PKC substrates(12) , we identified two PKC binding sequences: MARCKS-related protein (clone 35A) and a unique partial cDNA sequence with homology to adducins (clone 35H). Both were shown to be PKC substrates in vitro. In the present study, we report the complete coding sequence for 35H, identify it as a novel form of adducin, and establish that it is also a PKC substrate in vivo. These results underscore the utility of the PKC interaction cloning approach for identifying novel PKC substrates.
Figure 1:
Alignment of 35H with
- and
-adducins. Predicted amino acid sequences of 35H and
- and
-adducins were aligned using DNAsis (Hitachi) software.
Sequences containing 5 or more residues with >80% homology were
manually identified and shaded to easily identify conserved
domains.
In previous
studies, alignments of human -adducin with human and rat
-adducins revealed that the adducins share a homologous N-terminal
domain followed by a highly divergent region near the C terminus and a
highly conserved polybasic region at the extreme C terminus (residues
696-726 in
-adducin)(19, 20) . This overall
pattern is also apparent in the 35H alignment. Within the relatively
conserved N-terminal domains, divergent regions are separated by
stretches with near identity and conservative substitutions (Fig. 1, shaded regions). In general, there is a good
correlation in spacing of the variable and conserved domains within
-adducin,
-adducin, and 35H sequences. This alignment
suggests that adducins are a family of proteins with constant domains
separated at defined intervals by isoform-specific sequences, and
furthermore that 35H is a newly identified member of the adducin
family.
35H shares homology to - but not
-adducin in two
of the variable regions (437-447 and 472-484; numbers refer
to
-adducin residues shaded in Fig. 1). On the other hand,
35H shares homology to
-adducin in other variable regions
(8-15, 19-27, 85-92; numbers refer to
-adducin
residues shaded in Fig. 1). These results emphasize that 35H is
a unique sequence distinct from previously identified forms of adducin.
Both
-adducin and 35H diverge significantly from the
-adducin
putative calmodulin binding domain(446-454)(21) .
Diversity in this sequence suggests a potentially significant
difference in calmodulin regulation of 35H and
-adducin functions.
The conserved high positive charge density domains in the extreme C
termini are identical with the exception of the change of a potential
phosphorylatable Ser to Asn in 35H. 35H does not contain Tyr
of
-adducin or Arg
of
-adducin, which
have been identified as a potential tyrosine kinase phosphorylation
site or a determinant of cAMP-dependent kinase phosphorylation,
respectively(22) . Mutations in these sites have been linked to
hypertension.
Figure 2: Tissue distribution of 35H mRNA. Northern blots were hybridized with a radiolabeled 35H cDNA probe under stringent conditions. Blots shown were exposed to film for 24 h. Left, rat tissue mRNAs; right, human tissue mRNAs. H, heart; Br, brain; Sp, spleen; Lu, lung; Li, liver; Mu, muscle; Ki, kidney; Te, testes; Pl, placenta; Lu, lung; Pa, pancreas.
Figure 3:
Immunoblot analysis of renal 35H and
-adducin (clone 45). Homogenates were prepared from primary
growing renal proximal tubule epithelial cells (lane 1) and
rat kidney tissue (lane 2). Aliquots (100 µg of protein)
were separated on denaturing polyacrylamide gels and transferred to
nitrocellulose paper. Blots were stained with affinity-purified
anti-35H antibody (A) or with affinity-purified anti-45
antibody (B) at a concentration of 1 µg/ml. Results are
representative of more than five
experiments.
Figure 4:
Alignment of clone 45 and -adducin.
Predicted amino acid sequences of the open reading frame of the clone
45 partial cDNA and the corresponding region of human
-adducin
were aligned using DNAsis (Hitachi) software. Dots indicate
identical residues in the two sequences. Dashes indicate
gaps.
Figure 5:
Phosphorylation of 35H homologues in
intact primary growing RPTE. Primary proliferating RPTE were labeled
with P
. Samples from control (-) and
PDBu-treated (+) (200 nM PDBu for 10 min) cultures were
prepared. Cell lysates were immunoprecipitated with anti-35H antiserum,
nonimmune rabbit serum (non-i), or preimmune (pre-i)
serum (Ig). Immune complexes were separated on denaturing
polyacrylamide gels, and proteins were visualized by autoradiography.
Similar results were obtained in two additional
experiments.
Since -adducin is known to form
heterocomplexes with 120-kDa
-adducin(23) , we
hypothesized that the 120-kDa protein coprecipitating with 35H
antibodies may be
-adducin (our clone 45). To determine if
-adducin was present in the 35H immunecomplexes, 35H
immunoprecipitates of cell lysates were prepared and blotted to
nitrocellulose. Antibodies to 35H recognized the 80- and 90-kDa 35H in
the immunoprecipitates but did not recognize the 120-kDa phosphoprotein
apparent on the autorads (Fig. 6A). In contrast,
affinity-purified antibodies to clone 45 (
-adducin) fusion protein
recognized a 120-kDa band in the 35H immunoprecipitates (Fig. 6B). Since antibody to recombinant clone 45
protein does not recognize recombinant clone 35H protein and vice
versa (Fig. 7), these results strongly suggest that
-adducin is not directly precipitated with 35H antisera but that
it coprecipitates with the 35H proteins.
Figure 6:
Identification of an -adducin
homologue in 35H immunoprecipitates. Cell lysates were prepared from
primary growing RPTE and immunoprecipitated with nonimmune serum (lane 1) or 35H antiserum (lane 2). Immune complexes
were separated on denaturing polyacrylamide gels and transferred to
nitrocellulose paper. Left, the blot was stained with
affinity-purified anti-35H antibody. Right, the same blot was
cut and stained with affinity-purified anti-45 antibodies. Similar
results were obtained in two additional
experiments.
Figure 7: Specificity of anti-35H antibodies. Aliquots of affinity-purified 35H and 45 protein were dot-blotted onto nitrocellulose at the concentrations shown. Blots were stained with affinity-purified anti-35H antisera (A) or anti-45 antisera (B) at a concentration of 1 µg/ml.
Figure 8: Effects of PDBu on 35H subcellular distribution. Primary growing RPTEs were treated with PDBu (200 nM) for 0, 0.2, 1.5, or 16 h. Soluble (SOL) and cytoskeletal (CSK) fractions were prepared. Aliquots (50 µg of protein) were separated on denaturing polyacrylamide gels, blotted to nitrocellulose paper, and stained with affinity-purified anti-35H antibody at a concentration of 3 µg/ml. Similar results were obtained in two additional experiments.
PDBu-mediated changes in CSK 35H were more apparent by immunocytofluorescence than by immunoblot. In resting primary RPTE, 35H staining was concentrated in cell-cell junctions in detergent-extracted (CSK) preparations (Fig. 9). PDBu rapidly caused a decline in 35H staining concentrated at cell borders and an increase in staining in cytoplasmic dots (Fig. 9, B and C). The effect of PDBu was attenuated with prolonged PDBu treatment, which down-modulates PKC (Fig. 9D). Parallel studies with whole cell preparations indicated that the intensity and distribution of 35H staining were similar in CSK and whole cell preparations of resting cells (data not shown), consistent with the nearly quantitative recovery of 35H in the CSK fraction on immunoblots (Fig. 8). In whole cells, PDBu caused an increase in diffuse cytosolic staining and a decrease in cell border staining (data not shown) indicative of a redistribution from CSK to soluble fractions.
Figure 9: Immunocytochemical localization of 35H. Primary RPTE were grown on collagen-coated coverslips and treated with PDBu (200 nM) for the indicated times. Cells were extracted with Triton X-100, and the resulting cytoskeletal preparations were fixed and stained with affinity-purified anti-35H antibody followed by fluorescein-conjugated goat anti-rabbit antibody. PDBu treatment times were as follows: control (A), 0.2 h (B), 1.5 h (C), and 16 h (D).
The increase in soluble 35H (Fig. 8) and the decrease and
reorganization of CSK 35H (Fig. 9) observed after PDBu treatment
suggested that phosphorylation may regulate the distribution of 35H
proteins between soluble and CSK compartments. To test the hypothesis,
we immunoprecipitated 35H proteins from soluble and CSK fractions of P-labeled primary growing RPTE (Fig. 10). Basal
phosphorylation levels of 35H proteins in both soluble and CSK
fractions were low. PDBu stimulated phosphorylation of 80- and 90-kDa
35H proteins; these phosphoproteins were preferentially recovered in
the soluble fraction. Increased phosphorylation of the coprecipitating
120-kDa protein was also limited to the soluble pool. PDBu did not
stimulate phosphate incorporation into the CSK pool of these proteins.
Thus, although 35H protein mass was preferentially recovered in the CSK
fraction as demonstrated by immunoblots (Fig. 8), phosphorylated
forms were preferentially recovered in the soluble fraction (Fig. 10). Phosphorylation of 80- and 90-kDa 35H was attenuated
after 4-14 h of PDBu treatment, which correlates with the time
course for PKC down-modulation in these cells(25) .
Figure 10:
Subcellular localization of
phosphorylated 35H. Primary RPTE were labeled with 32P and
treated with 200 nM PDBu for the indicated times. Soluble (SOL) and cytoskeletal (CSK) fractions were prepared
and immunoprecipitated with anti-35H antiserum. Immune complexes were
separated on denaturing polyacrylamide gels and autoradiographed.
Similar results were obtained in two additional
experiments.
To ensure proper quantitation of blots, we compared antibody recognition of native and phosphorylated 35H. 35H bacterial fusion protein was phosphorylated with purified PKC in vitro(12) . 35H antibody detected both native and phosphorylated forms with the same intensity on immunoblots (data not shown). Thus, the increase in soluble 35H proteins in response to PDBu treatment cannot be attributed to increased antibody recognition of the phosphorylated form. Furthermore, we verified that 35H proteins were efficiently immunoprecipitated from both the soluble and CSK fractions (Fig. 11). Thus, preferential recovery of phosphorylated 35H homologues in the soluble fraction cannot be attributed to more quantitative recovery of 35H proteins in this fraction. In summary, our results demonstrate that in response to PDBu, 35H proteins are phosphorylated and redistributed from CSK to soluble fractions and that the phospho-35H proteins are preferentially recovered in the soluble fraction.
Figure 11: Immunoprecipitation of soluble and cytoskeletal 35H. Soluble (SOL) and cytoskeletal (CSK) fractions were prepared from primary growing RPTE. Fractions were immunoprecipitated with preimmune serum (p.i.) or 35H antiserum. Immune complexes and proportional aliquots of starting material (total) were separated on denaturing polyacrylamide gels, transferred to nitrocellulose, and stained with affinity-purified anti-35H antibody. Similar results were obtained in one additional experiment.
We have used a PKC overlay assay to detect PKC binding proteins and demonstrated a close correlation between PKC binding proteins (detected by blot overlay) and PKC substrates (detected by in vitro and in vivo phosphorylation assays)(9, 10, 11) . We have also used the overlay assay in an interaction cloning strategy to isolate cDNAs for PKC binding proteins(12) . Many, if not all, of the expressed cDNAs are PKC substrates in vitro. In the present studies, we have focused on clone 35H to determine if proteins corresponding to the cDNAs isolated according to PKC binding activity are substrates in vivo. Antibodies prepared to 35H recognize 80- and 90-kDa PKC substrates in RPTE cells, thus providing additional evidence that PKC binding proteins are PKC substrates. Furthermore, PKC phosphorylation regulated the CSK association of 35H (and clone 45) proteins. Thus, phosphorylation has demonstrable consequences on the function of these proteins, which, by analogy to other adducins, involves the regulation of actin assembly in the subcortical CSK.
Several lines of evidence
demonstrate that 35H is related to previously reported adducins. Like
- and
-adducins, 35H proteins are insoluble in Triton X-100
and concentrated at cell-cell junctions, and they are PKC
substrates(23, 26) . As previously reported for
-adducin(26) , phosphorylation of 35H proteins correlated
with their redistribution from CSK to soluble fractions. Furthermore,
-adducin (our clone 45) co-immunoprecipitates with 35H, indicating
that 35H proteins associate with
-adducin. Other evidence
emphasizes that despite the similarity to adducins, 35H is a unique
sequence. The alignment shown in Fig. 1indicates that 35H
diverges significantly from
- and
-adducins in discrete
regions. Northern analysis demonstrates that the size and tissue
distribution of 35H message are distinct from
- and
-adducins. Taken together, these data demonstrate that 35H is a
new member of the adducin family. We propose renaming it as
-adducin. Since
- and
-adducins are known to form
heterodimers, the structure and organization of adducins in cells and
tissues that express
-adducin but not
-adducin (e.g. kidney(20) ) have been questioned. It seems likely that
35H proteins (or other as yet unidentified adducins) could fulfill the
role of
-adducin in forming heterodimers with
-adducin in
these tissues.
Our original goal was to determine if sequences
isolated according to PKC binding in vitro were substrates in vivo. Several known PKC substrates were isolated in our
initial library screens for PKC binding proteins including
MARCKS-related protein (clone 35A), -adducin (clone 45), annexin
I, and annexin II. The fact that 35H, a unique sequence isolated
according to PKC binding activity, also appears to be a PKC substrate in vivo further confirms this conclusion. Additional studies
are in progress to determine if PKC is directly responsible for the
PDBu-stimulated 35H phosphorylation and if other kinases also
participate in 35H phosphorylation. In other studies, we have
determined that two other unique binding protein sequences (clones 72
and 35F) are PKC substrates in vitro and in vivo (data not shown). MARCKS, a major PKC substrate in many cells, is
also a PKC binding protein(9) . Thus, there is a strong
correlation between binding proteins detected in the overlay assay and
PKC substrates. It should be noted that in addition to their roles as
downstream targets and effectors of PKC activation, the apparent high
affinity between PKC and the binding proteins also allows for the
possibility that these sequences target PKCs to specific subcellular
addresses in vivo, although we do not yet have direct evidence
for this.
Each of the binding proteins we have tested binds phosphatidylserine (PS), and phosphorylation coordinately decreases PS and PKC binding(9, 11, 12) . Thus, PS bridging is an important component of PKC binding, and as described previously, it is also an important component for efficient trans-phosphorylation(27) . Thus, in our working model (Fig. 12), we propose that PKC and binding proteins are brought into close proximity due to their common affinity for PS. Since not all PS binding proteins are PKC binding proteins, additional protein-protein interactions appear to be required for formation of the high affinity ternary complex(28) . The model is drawn to indicate contact sites with the catalytic domain since the binding proteins are also substrates. PKC interactions with PS have been mapped to the regulatory domain, whereas MARCKS interactions with PS are due to electrostatic interactions with the high positive charge density domain containing the PKC phosphorylation site(8, 29, 30) .
Figure 12: Phosphorylation regulates ternary complex formation among PKCs, binding proteins, and phospholipid surfaces. PKC binding to substrates is dependent on the interactions of both PKC and the substrate with an anionic phospholipid surface such as PS (solid ribbon). PKC interactions with PS are mediated through PS binding sequences within the regulatory (Reg) domain, whereas substrate interactions with PS are mediated through electrostatic interactions with high positive charge density sequences (+++). The model is drawn to indicate that the substrates also interact with the catalytic (Cat) domain of PKC (in order for phosphorylation to occur). Phosphorylation coordinately attenuates the interaction between the substrate and both PS and PKC. In the case of MARCKS and 35H, decreased PS binding in vitro correlates with redistribution of the phosphorylated proteins from particulate to soluble fractions in vivo.
In addition to
regulating PKC binding and phosphorylation, PS binding also regulates
the association of MARCKS with cellular
membranes(29, 31) . In MARCKS and 35H(),
phosphorylated serine residues are located within high positive charge
density domains that support electrostatic interactions with the
negatively charged lipid surface. Thus, phosphorylation results in a
coordinate loss of both PKC and PS binding. Dissociation of PKC from
the binding protein represents a means for regulating the association
of PKC with its binding proteins/substrates (Fig. 12). Loss of
PS binding correlates with redistribution of binding proteins (such as
MARCKS and adducins) from particulate to soluble fractions. This
redistribution may be the means by which PKC alters the functions of
the CSK-associated proteins and exerts the profound changes in cell
shape elicited by phorbol ester treatment.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U35775[GenBank].