(Received for publication, October 2, 1995; and in revised form, December 27, 1995)
From the
The serine/threonine protein kinase PAK I (p21-activated protein kinase), a ubiquitous multipotential protein kinase of 58-60 kDa, has been shown to have cytostatic properties. Data from our laboratory show that PAK I is highly active in oocytes and quiescent and serum-starved cells, and injection of active PAK I into one blastomere of two-cell frog embryos inhibits cleavage of the injected blastomere. To clone the cDNA encoding PAK I, purified peptides from rabbit PAK I were sequenced, degenerate oligonucleotides were used to isolate PAK I clones from a rabbit spleen library, and the 5`-terminus was obtained by polymerase chain reaction. The entire cDNA sequence extends over 4471 nucleotides, with an open reading frame for a protein of 524 residues and a 3`-noncoding region of 2826 nucleotides. Clones with the same open reading frame but with 3`-noncoding regions of 1055 and 2478 nucleotides were isolated, suggesting the generation of different transcripts by alternative termination of transcription. The amino acid sequence of PAK I shows high homology to the p21-activated protein kinases from human placenta and rat brain and to yeast STE20. PAK I is activated by Cdc42(GTP). The PAK enzymes have been proposed to regulate the stress-activated protein kinase (also known as the Jun kinase) signaling pathway (Coso, O. A., Chiariello, M., Yu, J.-C., Teramoto, H., Crespo, P., Xu, N., Miki, T., and Gutkind, J. S.(1995) Cell 81, 1137-1146; Minden, A., Lin, A., Claret, F.-X., Abo, A., and Karin, M.(1995) Cell 81, 1147-1157).
The serine/threonine protein kinase PAK I (p21-activated protein
kinase) was first detected as an inactive holoenzyme that could be
converted into an active form by limited proteolysis with trypsin,
chymotrypsin, or a Ca-stimulated
protease(1, 2, 3, 4, 5, 6) .
Inactive PAK I is a monomer of 60 kDa, and the active peptide is 37
kDa(6) . PAK I appears to be highly conserved across species
and has been found in all animals and tissues examined to
date(1, 2, 3, 4, 5, 6) .
Two endogenously active forms of PAK I of 58 kDa were recently detected
in 3T3-L1 cells(5) , rabbit reticulocytes, (
)and
frog oocytes. (
)PAK I phosphorylates a number of proteins,
including histones H2B and H4(1) ; myosin light chain from
smooth muscle(3, 4) ; translational initiation factors
eIF-3, (
)eIF-4B, and eIF-4F(2, 7) ; and
avian and Rous sarcoma virus nuclear capsid protein
NC(8, 9, 10) .
PAK I activity is elevated
in quiescent and serum-starved cells, as opposed to actively dividing
cells. PAK I activity is also high in frog oocytes, but is rapidly
diminished following fertilization. Injection of
endogenously active or proteolytically activated PAK I into two-cell
frog embryos leads to cleavage arrest(11) . Other protein
kinases, including casein kinase II, protein kinase C, and the
catalytic subunit of cAMP-dependent protein kinase, have no effect on
cleavage.
The cloning and sequencing of the cDNA encoding PAK I, described herein, has revealed a mode of regulation of PAK I activity. The N-terminal regulatory domain contains a G protein-binding region similar to that of the G protein-activated protein kinases including rat brain PAK65(12) , human placenta PAK65(13) , and yeast STE20(14, 15) . Binding of the small G protein Cdc42 stimulates autophosphorylation and protein kinase activity, as shown by phosphorylation of H4.
For in situ CNBr cleavage, PAK I was subjected to electrophoresis on a 12.5% SDS-polyacrylamide gel (16) and transferred to an Immobilon-P membrane. PAK I was cleaved on the membrane with 4 mg/ml CNBr for 15 h at room temperature, and the peptides were eluted with 100 µl of 70% isopropyl alcohol and 1% trifluoroacetic acid for 3 h at room temperature, followed by 100 µl of 40% acetonitrile for 1 h at 37 °C. The extracts and the original cleavage solution were dried in a Speed Vac concentrator and separated by electrophoresis on a 16.5% SDS-polyacrylamide gel(16) . The peptides were transferred to a ProBlott membrane and subjected to amino acid sequence analysis. The N-terminal sequence of CNBr peptide 1 (20 kDa) was PEQWARLLGTSNXTKLEQKK, and that of CNBr peptide 2 (8 kDa) was XXXXFSTGGKDPLSANHXL.
To obtain the amino-terminal sequence of the p37 peptide, PAK I was partially digested with 4 µg/ml trypsin on ice for 1 min as described by Tahara and Traugh(1) , with the omission of bovine serum albumin. Peptides were separated by electrophoresis on a 12% SDS-polyacrylamide gel (16) and transferred to Immobilon-P, and the sequence was determined to be SVIDPIPAPVGDSHV.
For CNBr/endopeptidase Asp-N
cleavage, PAK I was cleaved with 10 mg/ml CNBr for 15 h at room
temperature, and the peptides were denatured, reduced, and
carboxymethylated as described(17) . The CNBr peptides were
digested with endopeptidase Asp-N, and the digest was acidified with
glacial acetic acid and separated by reverse-phase high pressure liquid
chromatography on a Vydac C column with a acetonitrile
gradient from 0 to 100%. Amino acid sequence analysis and mass
spectrometry were performed at the Biotechnology Instrumentation
Facility. The N-terminal sequences of six CNBr/endopeptidase Asp-N
peptides were as follows: CNBr/Asp-N peptide 1 (5497 Da),
DGFPSGAPALNTKVXETSAVVT; CNBr/Asp-N peptide 2 (4319 Da),
VEGEPPYLNENPLRALYLIAT; CNBr/Asp-N peptide 3 (3479 Da),
DVALGQECAIKQINLQKQPKKELIIN; CNBr/Asp-N peptide 4 (3479 Da),
DVEKRGSAKELLQHPF; CNBr/Asp-N peptide 5 (3226 Da),
DEXGIAAVXREXKQAKEFKGANQVIHR; and CNBr/Asp-N
peptide 6 (1438 Da), KELKNPNIVNF.
The effects of autophosphorylation on
activity were measured by preincubation of G protein with PAK I for 10
min as described above. A 20-µl sample was assayed in a final
volume of 70 µl containing phosphorylation buffer, 4.0 µg of
H4, and 0.2 mM [-
P]ATP (2000
dpm/pmol) by incubation for 15 min at 30 °C. Phosphorylation of H4
was analyzed by electrophoresis on a 15% SDS-polyacrylamide gel
followed by autoradiography (26) and quantified by
scintillation counting of the excised H4 band.
Following reverse transcription of mRNA from rabbit spleen and brain, degenerate oligonucleotides corresponding to CNBr/Asp-N peptides 4 and 5 were used to amplify a PAK I-specific polynucleotide. After 35 cycles of amplification, PCR products of 431 bp were subcloned and analyzed by DNA sequencing. The clones from spleen and brain were identical and contained the complete amino acid sequences of CNBr/Asp-N peptides 2, 4, and 5.
Figure 1: Strategy for cloning and sequencing of PAK I cDNA. A restriction map for the entire PAK I cDNA is shown at the top, and clones chosen for the complete sequence analysis by nested deletions are indicated below. Arrowheads show the directions and the positions sequenced by nested deletions. Boxes represent the open reading frame for PAK I, and solid lines represent untranslated regions. Endonuclease cleavage sites indicated are as follows: E, EcoRI; D, DraI; H, HindIII; P, PstI; S, SacI; X, XhoI. Sites in parentheses are within synthetic linkers.
The complete cDNA sequence of PAK I consists of 4471 bp, including a poly(A) tail of 18 nucleotides (Fig. 2). The cDNA sequence contains an open reading frame from nucleotides 74 to 1645, 73 nucleotides of 5`-untranslated region, and 2826 nucleotides of 3`-untranslated region. The 3`-untranslated regions of other clones isolated by screening of the rabbit spleen library were shorter than that of PAKI-4.1, but could be aligned without mismatches within the complete cDNA sequence and ended with poly(A) tails. The 3`-untranslated region of clone PAKI-7.3 was 1055 nucleotides, including a poly(A) tail of 20 nucleotides; those of clones PAKI-3.1 and PAKI-10.1 were 2478 nucleotides, including a poly(A) tail of 19 nucleotides, as indicated in Fig. 2. These results suggest that the different clones represent alternative transcription stops. The poly(A) addition signal for PAKI-7.3 could be AATAAT (20 nucleotides 5` of the poly(A) tail), and the poly(A) addition signal for PAKI-3.1/PAKI-10.1 could be AATTAAA (17 nucleotides 5` of the poly(A) tail) (Fig. 2). No sequence similar to the typical poly(A) addition signal (AATAAA) precedes the poly(A) tail of PAKI-4.1.
Figure 2:
Nucleotide and deduced amino acid
sequences of rabbit PAK I. The sequence of the coding strand of PAK I
cDNA is shown in a 5` to 3` orientation and extends over 4471
nucleotides, including 18 nucleotides of the poly(A) tail. The 3`-ends
of shorter clones, including PAKI-7.3 at nucleotide 2698 and
PAKI-3.1/PAKI-10.1 at nucleotide 4122, which terminated with poly(A)
tails, are indicated (⇑). Possible poly(A) addition signals are underlined. The deduced amino acid sequence is shown below the
nucleotide sequence in single-letter code. It consists of 524 amino
acid residues. Amino acid sequences determined by microsequencing of
PAK I peptides are underlined. From the N terminus to the C
terminus, the peptides are as follows: CNBr peptide 2, CNBr peptide 1,
CNBr/Asp-N peptide 1, p37, CNBr/Asp-N peptide 3, CNBr/Asp-N peptide 6,
CNBr/Asp-N peptide 5, CNBr/Asp-N peptide 2, and CNBr/Asp-N peptide 4.
The N-terminal amino acid residue of the proteolytically activated p37
fragment of PAK I is shown (first ). Also shown is the
start of the catalytic domain (second
) as determined by
comparison with the sequence of other protein kinases (31, 32) .
The
deduced amino acid sequence of the open reading frame encodes a
polypeptide of 524 amino acid residues with a calculated molecular mass
of 58,027 Da. All nine partial amino acid sequences, obtained by
microsequence analysis of purified peptides of PAK I, could be aligned
within the deduced amino acid sequence (Fig. 2). The peptide
beginning with SVID at residue 197 indicates the N terminus of the p37
peptide generated by limited trypsin digestion, which contains the
catalytic domain. The beginning of the catalytic domain at residue 247
was determined by comparison with the sequences of other protein
kinases using the alignment of Hanks et
al.(27, 28) . Peptide p37 contains part of the
regulatory domain (from residues 197 to 246) and all 11 conserved
subdomains characteristic of the catalytic domain of protein kinases.
The GXGXXG motif involved in nucleotide binding
begins at residue 256; residue 278 is the invariant lysine that
contacts the - and
-phosphates of the ATP; residue 368 is the
aspartate that acts as a general base for the removal of a proton from
the hydroxyl group of the protein substrate; residue 370 is the lysine
that binds to the
-phosphate of the ATP; and residue 386 is the
aspartate that is part of the conserved DFG motif and chelates the
Mg
bound to the ATP. A putative binding site for
Rho-like G proteins was identified in the regulatory domain between
residues 73 and 107 by sequence comparison with PAK65 from rat brain
and human placenta and yeast
STE20(12, 13, 14, 15) .
Amino acid sequence alignment of the total proteins revealed 95% identity between rabbit PAK I and human PAK65 and 78% identity between rabbit PAK I and rat PAK65 (Fig. 3). Within the catalytic domain, rabbit PAK I has 99% identity with human PAK65, 92% with rat PAK65, and 65% with yeast STE20. In the regulatory domain, PAK I has significantly less homology with STE20 and rat PAK65, except for the G protein-binding region. Compared with rat PAK65, PAK I was 20 residues shorter because of five gaps in the regulatory domain ranging from 1 to 10 residues. Human PAK65 was 18 residues shorter than rabbit PAK I because of differences at the N terminus over the first 30 amino acid residues; the rest of the amino acid sequence was very similar to that of rabbit PAK I.
Figure 3: Multiple sequence alignment of p21-activated protein kinases. The deduced amino acid sequences of rabbit PAK I, human PAK65, rat PAK65, and the GTP/p21-binding and catalytic domains of yeast STE20 were aligned using the programs Pileup, Lineup, and Pretty of the University of Wisconsin Genetics Computer Group Package Version 8(25) . The consensus sequence indicates identical residues for at least two of the protein sequences. Deviations from the consensus sequence are indicated by lower-case letters; gaps are indicated by dots. The positions of the 11 conserved subdomains of the catalytic domain of the protein kinases are shown below the alignment in Roman numerals.
Figure 4:
G protein-stimulated autophosphorylation
of PAK I. PAK I (0.1 µg) was incubated with
[-
P]ATP for the times indicated, alone or
in the presence of the GST fusion protein of Rac1, Cdc42Hs, or RhoA
bound to GTP
S. Autophosphorylation was analyzed by
SDS-polyacrylamide gel electrophoresis followed by autoradiography. The
autoradiogram is shown.
To examine the effects of autophosphorylation on activation
of PAK I, protein kinase activity was measured by phosphorylation of
the substrate H4 (Fig. 5). PAK I was incubated under
autophosphorylation conditions for 10 min, either alone or with
GST-Rac1, GST-Cdc42Hs, or GST-RhoA preloaded with GTPS or GDP, and
then assayed with H4. Phosphorylation of H4 was dependent on the level
of autophosphorylation of PAK I. Without addition of G protein, a low
level of autophosphorylation and PAK I activity with H4 was observed,
which was subtracted from the data obtained in the presence of G
protein. Addition of GST-Cdc42Hs(GTP
S) resulted in an increased
autophosphorylation of PAK I of 0.97 mol/mol and a stimulation of the
rate of phosphorylation of H4 of 18,261 pmol/min/mg. With
GST-Cdc42Hs(GDP), stimulation of autophosphorylation was 0.12 mol/mol,
and the activity was 2154 pmol/min/mg. These values were
8-fold
lower than those observed with Cdc42Hs(GTP
S). GST-Rac1(GTP
S)
resulted in only a modest stimulation,
10-fold lower than that
observed with Cdc42Hs(GTP
S); autophosphorylation and activity were
0.09 mol/mol and 1515 pmol/min/mg, respectively. GST-RhoA(GTP
S)
had no stimulatory effect on either autophosphorylation or activity.
Figure 5:
Stimulation of PAK I activity by
autophosphorylation. PAK I (0.1 µg) was autophosphorylated for 10
min in the presence of the GST fusion protein of Cdc42Hs, Rac1, or RhoA
preloaded with GTPS or GDP. Following autophosphorylation, assays
with H4 were carried out and analyzed on a 15% SDS-polyacrylamide gel,
followed by autoradiography and scintillation counting of the excised
H4 bands. Phosphorylation of H4 in the absence of G protein was used as
background and subtracted from the data.
The cDNA isolated by screening of a rabbit spleen cDNA library and by 5`-RACE-PCR using rabbit spleen mRNA contains the information for the complete sequence of PAK I. The deduced amino acid sequence contains the catalytic domain and an N-terminal regulatory domain, including a region shown previously to bind Rho-like G proteins. All of the partial amino acid sequences determined by microsequencing of nine peptides from PAK I could be aligned with the deduced amino acid sequence. The calculated molecular mass of 58,027 Da is in accordance with the apparent molecular mass of 58-60 kDa displayed by native PAK I on SDS-polyacrylamide gels(5, 6) .
The cDNA for PAK I from rabbit spleen contains a long 3`-untranslated region of 2826 nucleotides. Long 3`-untranslated regions are characteristic of highly regulated genes and are involved in the stability and translational efficiency of the mRNA(31, 32) . The isolation of PAK I clones from rabbit spleen with shorter 3`-untranslated regions of 1055 and 2478 nucleotides, including the poly(A) tails, suggests the existence of different transcripts for PAK I generated by alternative transcription termination and polyadenylation sites. Transcripts of PAK I with 3`-untranslated regions of different lengths could have different stability and/or translation efficiency. Therefore, the amount of PAK I protein could be regulated in part by the expression of transcripts with 3`-untranslated regions of different lengths.
Sequence
comparison shows high sequence homology to the yeast protein kinase
STE20 (14, 15) and PAK65 from rat brain and human
placenta(12, 13) , suggesting that they belong to a
family of related protein kinases. Rat PAK65 appears to be a
brain-specific isoform; a protein with the corresponding molecular mass
was detected only in brain extracts, but not in other tissues, by
overlay assays with radiolabeled Rac1(GTP) and Cdc42Hs(GTP) and by a
specific antibody raised against the N-terminal region of rat brain
PAK65(12, 13) . Unlike brain-specific rat PAK65,
inactive PAK I was purified from a number of tissue and cell types,
including rabbit reticulocytes, liver, and skeletal muscle; chicken
gizzard, liver, and brain; bovine liver; mouse 3T3-L1
cells(1, 2, 3, 4, 5, 6) ;
and frog oocytes and embryos. The protein reacted with
antibodies raised against PAK I from rabbit reticulocytes. PAK I from
rabbit spleen and PAK65 from human placenta have a high degree of
sequence homology, but the N-terminal 30 amino acid residues are
completely different. Human PAK65 lacks 18 amino acid residues, and the
remaining 12 amino acid residues cannot be aligned with either rabbit
PAK I or rat PAK65. Therefore, it is unlikely that the two protein
kinases are homologous enzymes, but they appear to be closely related
isoforms.
PAK65 from rat brain and human placenta as well as two additional PAK enzymes detected in human neutrophils were shown to be autophosphorylated upon binding of the G proteins Rac1 and Cdc42, but not RhoA; autophosphorylation resulted in activation as shown by stimulation of phosphorylation of the universal substrate, myelin basic protein(12, 13, 29, 30) . Binding of the GTP-bound form of GST-Cdc42Hs, but not GST-Rac1 or GST-RhoA, greatly induced autophosphorylation of PAK I from rabbit reticulocytes, and autophosphorylation resulted in activation of the protein kinase.
STE20 in budding yeast plays a key role in signal transduction
pathways connecting membrane receptors with the MAP kinase
cascade(33) . In the mating differentiation pathway, a
pheromone initiates differentiation by activation of STE20, which
activates the MAP kinase cascade. STE20 and MAP kinases are also
involved in other signal transduction pathways in yeast, including the
invasive growth response and pseudohyphal development pathways
initiated by nutritional starvation. Different isoforms of STE20 and
MAP kinases could have alternative functions in regulating different
signal transduction pathways(33) . PAK I and other PAK enzymes,
as the mammalian counterparts of yeast STE20 protein kinases, also
appear to be involved in the coordination of signal transduction
pathways. It has recently been hypothesized (34, 35) that the Rac1/Cdc42-regulated PAK enzymes are
a key point regulator of the stress-activated protein kinase or Jun
kinase signaling pathway, which regulates c-Jun activity. Biochemical
evidence from our laboratory suggests that PAK I is involved in
regulation of cytostasis as shown by the high level of activity in
quiescent and serum-starved cells and lower levels of PAK I activity in
actively dividing cells. PAK I activity is also high in
mature frog oocytes, but is greatly diminished following
fertilization.
Injection of active PAK I into two-cell
embryos has been shown to inhibit cell division in the injected
blastomere(11) . Thus, PAK I appears to be a candidate for
regulation of the signaling cascade that is activated in response to
stress.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U46915[GenBank].