(Received for publication, December 26, 1995; and in revised form, February 1, 1996)
From the
PKN is a fatty acid-activated serine/threonine kinase that has a catalytic domain highly homologous to that of protein kinase C in the carboxyl terminus and a unique regulatory region in the amino terminus. Recently, we reported that the small GTP-binding protein Rho binds to the amino-terminal region of PKN and activates PKN in a GTP-dependent manner, and we suggested that PKN is located on the downstream of Rho in the signal transduction pathway (Amano, M., Mukai, H., Ono, Y., Chihara, K., Matsui, T., Hamajima, Y., Okawa, K., Iwamatsu, A., and Kaibuchi, K.(1996) Science 271, 648-650; Watanabe, G., Saito, Y., Madaule, P., Ishizaki, T., Fujisawa, K., Morii, N., Mukai, H., Ono, Y. Kakizuka, A., and Narumiya, S.(1996) Science 271, 645-648). To identify other components of the PKN pathway such as substrates and regulatory proteins of PKN, the yeast two-hybrid strategy was employed. By this screening, a clone encoding the neurofilament L protein, a subunit of neuron-specific intermediate filament, was isolated. The amino-terminal regulatory region of PKN was shown to associate with the head-rod domains of other subunits of neurofilament (neurofilament proteins M and H) as well as neurofilament L protein in yeast cells. The direct binding between PKN and each subunit of neurofilament was confirmed by using the in vitro translated amino-terminal region of PKN and glutathione S-transferase fusion protein containing the head-rod domain of each subunit of neurofilament. PKN purified from rat testis phosphorylated each subunit of the native neurofilament purified from bovine spinal cord and the bacterially synthesized head-rod domain of each subunit of neurofilament. Polymerization of neurofilament L protein in vitro was inhibited by phosphorylation of neurofilament L protein by PKN. The identification and characterization of the novel interaction with PKN may contribute toward the elucidation of mechanisms regulating the function of neurofilament.
We have reported a novel serine/threonine protein kinase,
designated PKN, having a catalytic domain homologous to the PKC ()family and unique amino-terminal
sequences(1, 2) . The amino-terminal region of PKN
contains repeats of a leucine zipper-like motif, suggesting promotion
of protein-protein association through hydrophobic
interactions(3) , and the basic region adjacent to the first
leucine zipper-like motif, which is conserved through evolution in
vertebrates (4) and among the various isoforms of
PKN(5) . In the previous study, we demonstrated that truncation
of the amino-terminal region of PKN by limited proteolysis results in
the generation of the catalytically active form in vitro, and
unsaturated fatty acid and some detergent can remove restriction of the
catalytic activity by its amino-terminal region at relatively low
concentrations in vitro(1, 6) . Thus, regions
contained in the amino-terminal portion of PKN are presumed to be
critical for the regulation of the biological activity of this enzyme.
Recently, we demonstrated that Rho, a small GTP-binding protein, binds to PKN in a GTP-dependent fashion and that this binding leads to the activation of PKN(7, 8) , suggesting that PKN is one of the targets of Rho. Several reports have been accumulated showing that multiple protein-protein interactions within the regulatory region of serine/threonine protein kinases are important for the regulation of the enzyme activities in vivo, such as Raf-1 and p21-activated protein kinase(9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19) . For example, current models of Raf-1 activation suggest that this kinase is bound in a native complex with 14-3-3 proteins (10, 11) and p50 and hsp90 proteins(12, 13) . Raf-1 also binds to Ras in a GTP-dependent fashion (14, 15, 16, 17) , and active Ras is thought to recruit Raf-1 to the plasma membrane, where it interacts with other modulators of its activity and relevant substrates(18, 19) . The binding site on Raf-1 for 14-3-3 proteins is distinct from the Ras binding domain, and Raf-1 molecules bound to 14-3-3 proteins may simultaneously bind to Ras(10, 11) . In our preliminary experiment, PKN immunoreactivity in the crude extract from rat brain was eluted broadly on gel filtration, suggesting that PKN formed complexes. Thus, in the analogy with Raf-1, we attempted to identify other proteins that interact with PKN and potentially regulate its activity by using a yeast two-hybrid system, with the amino-terminal regulatory region of PKN as a target protein. By this screening, a cDNA clone encoding the head-rod domain of NFL, a subunit of neuron-specific intermediate filament protein, was isolated from a human brain cDNA library. In this report we characterize the interaction between PKN and each subunit of NF and vimentin and raise the possibility that PKN can play a role in the regulation of assembly of NF and provide a potential junction from signals generated by growth factor stimulation to intermediate filaments.
Figure 1: Fusion constructs for each subunit of NF used in this study. The full sequence of each subunit of NF is represented by a gray box. Thick boxes indicate the approximate location of the rod domain of each subunit of NF. The VP16 transactivation domains (cross-hatched boxes), LexA DNA binding domains (hatched boxes), and glutathione S-transferase (solid boxes) were fused to the various deletion mutants of NF (open boxes). The dashed box indicates the deleted sequence. The original clone isolated from the library pGAD10-NFL#21 is shown at the bottom of the figure. Restriction sites: T, Tth111I; X, XhoI; P, PstI; B, BglII; K, KpnI.
Other yeast expression vectors were prepared as follows. The yeast expression vector pVP/NFL#21, for VP16ad fused to the head-rod domain of human NFL, was made by subcloning an EcoRI insert (amino acids 1-349) of the library plasmid 21 originally isolated from two-hybrid screening into pVP16 vector. Vectors for VP16ad fused to the tail domain of NFL, VP16ad fused to the tail domain of NFH, VP16ad fused to the head-rod domain of NFM, and VP16ad fused to the tail domain of NFM were constructed by subcloning each BamHI/NotI insert of pGST/NFLt, pGST/NFHt, pGST/NFMhr, and pGST/NFMt (described later under ``Preparation of GST Fusion Proteins''), respectively, into pVP16. pVP/NFHf, for VP16ad fused to the full length of NFH, was constructed by digesting pBH, a plasmid containing the entire coding region of NFH in pBluescript II SK vector (kindly provided by Dr. N. Hirokawa, University of Tokyo), with BglII, and the cDNA insert was subcloned into pVP16. pVP/NFHhr for VP16ad fused to the head-rod domain of NFH was made by digesting pVP/NFHf with Tth111I/EcoRI and filling in the ends with T4 DNA polymerase and ligating them together with T4 DNA ligase. This removed the DNA sequences 3`-terminal to the Tth111I site, which encodes the tail domain of NFH. pBTM/NFL#21 and pBTM/NFLt for LexAbd fused to the head-rod domain of NFL and LexAbd fused to the tail domain of NFL, respectively, were constructed as described above except for using pBTM116 instead of pVP16. pBTM/PKNN1 for LexAbd fused to PKNN1 and pVP/PKNN1 for VP16ad fused to PKNN1 were constructed by subcloning the EcoRI/BamHI fragment of human PKN into pBTM116 and pVP16, respectively. pBTM/PKNC1 for LexAbd fused to the carboxyl-terminal region of PKN (amino acids 511-942, this region was designated as PKNC1) and pVP/PKNC1 for VP16ad fused to PKNC1 were constructed by subcloning the ClaI/EcoRI fragment of human PKN into pBTM116 and pVP16, respectively.
Expression and purification of GST or GST fusion proteins were performed according to the manufacturer's instruction (Pharmacia Biotech Inc.) The eluate from glutathione-Sepharose 4B (Pharmacia Biotech Inc.) was dialyzed overnight against 10 mM Tris/HCl at pH 8.8 containing 1 mM EDTA, 1 mM DTT, and 0.1 µg/ml leupeptin.
Figure 2:
Interaction between portions of PKN and NF
in the two-hybrid system. For each transformation, five independent
colonies were picked from nonselected (His+) plates, patched, and
grown for 3 days before they were tested for production of
-galactosidase. The data are representative of these colonies and
three independent transformations. Panel A, interaction
between PKN and NFL. Plasmids for VP16ad (pVP16; lines 7 and 10), VP16ad fused to the head-rod domain of NFL (pVP/NFL#21; lines 1-3), VP16ad fused to the tail domain of NFL
(pVP/NFLt; lines 4-6), VP16ad fused to PKNN1 (pVP/PKNN1; lines 8 and 11), and VP16ad fused to PKNC1
(pVP/PKNC1; lines 9 and 12) were transformed into
yeast with the plasmids for LexAbd (pBTM116; lines 1 and 4), LexAbd fused to the head-rod domain of NFL (pBTM/NFL#21; lines 7-9), LexAbd fused to the tail domain of NFL
(pBTM/NFLt; lines 10-12), LexAbd fused to PKNN1
(pBTM/PKNN1; lines 2 and 5), and LexAbd fused to
PKNC1 (pBTM/PKNC1; lines 3 and 6). Panel B,
interaction between the amino-terminal region of PKN and each subunit
of NF. Plasmids for VP16ad fused to the head-rod domain of NFL
(pVP/NFL#21; line 1), VP16ad fused to the head-rod domain of
NFM (pVP/NFMhr; line 2), VP16ad fused to the head-rod domain
of NFH (pVP/NFHhr; line 3), VP16ad fused to the tail domain of
NFL (pVP/NFLt; line 4), VP16ad fused to the tail domain of NFM
(pVP/NFMt; line 5), and VP16ad fused to the tail domain of NFH
(pVP/NFHt; line 6) were transformed into yeast with LexAbd
fused to PKNN1 (pBTM/PKNN1; lines
1-6).
Figure 3:
In vitro association of PKN with
each subunit of NF. S-Labeled in vitro translated
PKN was incubated with bacterially synthesized GST or GST-fused NF
proteins. GST or GST-fused proteins were collected with
glutathione-Sepharose beads (G-beads), analyzed by SDS-PAGE, and
followed by autoradiography as described under ``Experimental
Procedures.'' Aliquots of the initial binding reaction mixtures
(10 µl) were removed before precipitation and applied to
electrophoresis (Input). For reference, molecular mass markers
are indicated in kDa at the left. Shown are typical results
from three independent experiments. Panel A, interaction
between PKN and various deletion mutant of NFL. The amino-terminal
region of PKN (PKNN2) or the carboxyl-terminal region of PKN (PKNC2)
was incubated with GST (lanes 4, 6, 10, and 12) or GST fused to various deletion fragments of NFL
(GST/NFL#21, lanes 1, 5, 7, and 11;
GST/NFLdelA, lanes 2 and 8; GST/NFLdelB, lanes 3 and 9). Panel B, interaction between the
amino-terminal region of PKN and the head-rod domain of each subunit of
NF. PKNN2 was incubated with GST (lanes 4 and 8) or
GST fused to the head-rod domain of NFL (GST/NFL#21, lanes 1 and 5), the head-rod domain of NFM (GST/NFMhr, lanes
2 and 6), and the head-rod domain of NFH (GST/NFHhr, lanes 3 and 7). The position of labeled protein is
indicated by a white arrowhead.
We reported previously that RhoA binds to the amino-terminal region of PKN. Given the interaction of NFL with the amino-terminal regulatory region of PKN, it was possible that this protein interacts with the Rho binding domain of PKN and might compete with Rho for binding to PKN. In an in vitro binding assay, bacterially synthesized NFL did not compete with the bacterially synthesized RhoA for the binding to the amino-terminal region of PKN (data not shown).
Since NFL bound to the regulatory region of PKN, a possibility was raised that NFL affects the kinase activity of PKN. To test this possibility, we added various concentrations of the bacterially synthesized NFL to the PKN assay mixture and examined the phosphorylation activity using a synthetic peptide based on the pseudosubstrate site of PKC as a substrate(1) . We found no obvious effect of NFL on the peptide phosphorylation activity of PKN (data not shown).
Figure 4: Phosphorylation of NF by PKN. Panel A, phosphorylation of NF in the presence or absence of arachidonic acid. a and b, protein silver staining and autoradiograph of SDS-PAGE of the purified NF proteins incubated with purified rat PKN for 5 min at 30 °C with (lane 2) or without (lane 1) 40 µM arachidonic acid. NFH, NFM, and NFL are indicated beside lane 2 by H, M, and L, respectively. The position of autophosphorylation of PKN is indicated by a white arrowhead. Panel B, time course of phosphorylation of the isolated NF containing triplet proteins by PKN. Shown are the autoradiographs of SDS-PAGE of the dephosphorylated form (lanes 1-5) and phosphorylated form (lanes 6-10) of NF incubated with rat purified PKN and 40 µM arachidonic acid for 0 min (lanes 1 and 6), 10 min (lanes 2 and 7), 30 min (lanes 3 and 8), 60 min (lanes 4 and 9), and 120 min (lanes 5 and 10) at 30 °C. NFL, NFM, and NFH are indicated beside lanes 5 and 10 by L, M, and H, respectively. The positions of autophosphorylation of PKN are indicated by white arrowheads. Molecular mass markers are indicated in kDa at the left. Panel C, the amount of label incorporated into each NF protein. Open circles, NFL pretreated with alkaline phosphatase (indicated by dL); closed circles, NFL untreated with alkaline phosphatase (indicated by L); open triangles, NFH pretreated with alkaline phosphatase (indicated by dH); closed triangles, NFH untreated with alkaline phosphatase (indicated by H); open squares, NFM pretreated with alkaline phosphatase (indicated by dM); closed squares, NFM untreated with alkaline phosphatase (indicated by M). The data are representative of three independent experiments. Panel D, the effect of dephosphorylation on the electrophoretic mobility of NF protein. Shown is protein staining with Coomassie Blue of native NF (lane 1) and NF pretreated with alkaline phosphatase (lane 2). Molecular mass markers are indicated in kDa at the left.
To
examine the phosphorylation site of NF by PKN, we prepared bacterially
synthesized GST fused to the head-rod domain and GST fused to the tail
domain of each subunit of NF and subjected them to an in vitro phosphorylation assay. As shown in Fig. 5, P
was incorporated to the head-rod domain of each NF in the ratio of
3:10:2 for NFH:NFM:NFL. The GST fused to the tail domain of each
subunit was not labeled at all. PKN also phosphorylated the head-rod
domain of vimentin (data not shown). This clearly indicated that
phosphorylation sites were located exclusively in the head-rod domain
of these intermediate filaments.
Figure 5: Identification of phosphorylation sites at the head-rod domain of each subunit of NF. Panels A and B, protein staining and autoradiograph of SDS-PAGE of GST-fused NF proteins phosphorylated by PKN, respectively. GST fused to the head-rod domain of NFH (GST/NFHhr; lane 1), GST fused to the head-rod domain of NFM (GST/NFMhr; lane 3), GST fused to the head-rod domain of NFL (GST/NFL#21; lane 5), GST fused to the tail domain of NFH (GST/NFHt; lane 2), GST fused to the tail domain of NFM (GST/NFMt; lane 4), and GST fused to the tail domain of NFL (GST/NFLt; lane 6) were incubated with PKN purified from rat brain and 40 µM arachidonic acid for 10 min at 30 °C as described under ``Experimental Procedures.'' The positions of the GST fused to the tail domain of NFL, GST fused to the tail domain of NFM, and GST fused to the tail domain of NFH are indicated beside lane 6 of panel A by L, M, and H, respectively. The position of autophosphorylation of PKN is indicated by a white arrowhead in panel B. The positions of the head-rod domain of GST fused to each subunit of NF are indicated by black arrowheads.
We investigated
whether NFL polymerizes in an in vitro binding analysis. The
bacterially produced GST fused to the head-rod domain of NFL or GST
fused to the full length of NFL was mixed with in vitro translated NFL in buffer at pH 8.5 with 1 mM MgCl. Then the pH of the reaction mixture was shifted
to 7.2 and incubated for 1 h at 35 °C. After extensive washing,
proteins bound to the beads were analyzed by autoradiography. As shown
in Fig. 6, polymerization of NFL was clearly detected. Next, we
determined whether phosphorylation of NFL by PKN inhibits the
polymerization of NFL in this assay system. The GST fused to the
head-rod domain of NFL and GST fused to the full length of NFL were
phosphorylated by PKN, transferred to the reaction mixture, and mixed
with in vitro translated NFL. As shown in Fig. 6, the
binding of the head-rod domain and the full length of NFL to in
vitro translated NFL was very weakly detected, indicating that
phosphorylation of NFL by PKN inhibited the polymerization of NFL.
Figure 6:
Effect of phosphorylation on the
polymerization of NFL. S-Labeled in vitro translated NFL and PKN purified from rat testis were incubated
with bacterially synthesized GST (lanes 5 and 10),
GST fused to the head-rod domain of NFL (GST/NFL#21; lanes 1, 2, 6, and 7), or GST fused to the full
length of NFL (GST/NFLf; lanes 3, 4, 8, and 9) with (lanes 2, 4, 7, and 9) or without (lanes 1, 3, 6, 8) 100 µM ATP. GST or GST-fused proteins were
collected with G-beads, analyzed by SDS-PAGE, and followed by
autoradiography as described under ``Experimental
Procedures.'' The position of the labeled protein is indicated by
a white arrowhead. Molecular mass markers are indicated in kDa
at the left. Shown are typical results from three independent
experiments.
Recently, increasing evidence has accumulated to suggest that
continuous turnover of intermediate filaments is involved in
maintaining the intermediate filament structure and function in living
cells. Transfection of native and mutated intermediate filament genes
or microinjection of biotin-labeled intermediate filaments has revealed
the successive incorporation of newly synthesized or microinjected
intermediate filament proteins into preexisting intermediate filament
networks(32, 33, 34, 35, 36, 37) and
the disruption of endogenous intermediate filament networks by the
integration of assembly-incompetent mutant
proteins(38, 39, 40) . NF, a neuronal
intermediate filament, was also reported to have a dynamic structure,
which turns over within a small region of the axoplasm by exchanging
subunits at discrete sites on the filaments(41) . One of the
possible mechanisms of the growth-dependent regulation of NF turnover
is the phosphorylation of NF triplet proteins. Protein kinases
responsible for the phosphorylation of NF subunits in the intact tissue
are yet unclear. Several laboratories have directed their attention to
phosphorylating activities that remain bound to vertebrate NFs through
purification
procedures(42, 43, 44, 45) . Wible et al.(46) reported the isolation of a NF-associated
protein kinase of 67 kDa from bovine spinal cord which phosphorylates
NF. PKN is clearly distinguished from this protein kinase by molecular
weight, by their marked preference for the tail domain of NFH, and by
their lack of autophosphorylation activity. Protein kinases that
phosphorylate all three subunits of NF were partially purified by
several other laboratories, although their molecular weights and
structural characteristics are unknown. One of these, partially
purified by Toru-Delbauffe and Pierre (47) and Toru-Delbauffe et al.(48) , has a high specificity for NFH and is
insensitive to 5 mM Ca. Another one,
purified by Dosemeci et al.(49) , has marked
preference for casein rather than histone or NF as substrate. Another,
purified by Hollander and Bennett(50) , phosphorylates
efficiently the carboxyl-terminal tail domain of NFM. Judging from
these characteristics, PKN seems to be different from these kinases.
Recently, Xiao and Monteiro (51) detected four prominent
kinases (NAKs) with molecular masses of 115, 95, 89, 84 kDa by in
situ gel kinase assay in an affinity-purified fraction by using
recombinant NFH from mouse brain extract(51) . The 115-kDa
molecular mass of NAK is close to that of human PKN, which is120
kDa(2) . These investigators reported that NAKs phosphorylate
all three NF subunits and that partial dephosphorylation of NF with
alkaline phosphatase does not affect the phosphorylation of the NFH and
NFM by NAKs. Thus PKN is similar in this respect. However, PKN bound to
the head-rod domain of NFH, and NAKs were found on the basis of their
retention by the tail domain of the NFH affinity column. Purification
and information about molecular aspects of NAKs are expected. Dosemeci
and co-workers (49, 52) reported that bovine NF
preparation contains known Ca
/calmodulin-dependent
kinase, PKC, and PKA as well as activator-independent kinase
activities. In vitro phosphorylation of NF at the
amino-terminal domain by PKA or PKC inhibits the assembly process of NF
and induces disassembly of preexisting filaments(53) . In the
present study, we revealed that PKN phosphorylated NF at the
amino-terminal domain, was able to inhibit the assembly of NFL in
vitro, and that PKN was another candidate for being a regulator of
NF assembly.
We reported here that the regulatory domain of PKN binds to the head-rod domain of NF and vimentin. What is the meaning of this association? Although additional evidence is required for determining whether PKN phosphorylates NF in vivo, one possible explanation of the interaction between the regulatory domain of PKN and NF is that an association interface outside the catalytic domain is required for substrate recognition and could serve to target PKN to physiologically important substrates. Precedence for protein association outside the conserved catalytic domain of protein kinase as an important targeting mechanism during signal transduction is provided by studies of Src homology domains (54, 55, 56, 57) and the regulatory region of PKC(58, 59) . In our preliminary experiment, some clones that were isolated by this two-hybrid screening were indicated to be phosphorylated in vitro by PKN. It is possible that PKN uses its regulatory domain as an association domain for all physiologically important substrates. An alternative possibility is that the binding of PKN to one substrate serves to promote the phosphorylation of other proteins in the same complex or that NF acts only as an adaptor that promotes the activity of PKN toward adjacent specific cytoskeletal targets.
We recently found that PKN directly
binds to the GTP-bound form of Ras-related small GTPase Rho and is
activated by this binding both in a cell-free system and in cultured
cells(7, 8) . Rho works as a molecular switch in
diverse cellular processes such as stimulus-evoked cell
adhesion(60, 61, 62) , cell
motility(63) , regulation of smooth muscle
contraction(64) , and cytokinesis(65, 66) .
Rho was presented to exert these actions by inducing certain types of
cytoskeletal structures(60, 65, 66) .
Paterson et al.(67) reported the collapse of the
intermediate filament into irregular thick bundles after microinjection
of constitutive activated Rho (ValRho) into subconfluent
Swiss 3T3 cells; however, the biochemical basis of the Rho effect is
not known. From the data presented in this paper, PKN, positioned in
the downstream of Rho signal, might regulate the assembly-disassembly
of intermediate filaments.