The Department for Cell and Molecular Biology, University of Umeå, S-901 87 Sweden
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Abstract |
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Oncoprotein 18 (Op18, also termed p19, 19K, metablastin, stathmin, and prosolin) is a recently identified regulator of microtubule (MT) dynamics. Op18 is a target for both cell cycle and cell surface receptor-coupled kinase systems, and phosphorylation of Op18 on specific combinations of sites has been shown to switch off its MT-destabilizing activity. Here we show that induced expression of the catalytic subunit of cAMP-dependent protein kinase (PKA) results in a dramatic increase in cellular MT polymer content concomitant with phosphorylation and partial degradation of Op18. That PKA may regulate the MT system by downregulation of Op18 activity was evaluated by a genetic system allowing conditional co-expression of PKA and a series of kinase target site-deficient mutants of Op18. The results show that phosphorylation of Op18 on two specific sites, Ser-16 and Ser-63, is necessary and sufficient for PKA to switch off Op18 activity in intact cells. The regulatory importance of dual phosphorylation on Ser-16 and Ser-63 of Op18 was reproduced by in vitro assays. These results suggest a simple model where PKA phosphorylation downregulates the MT-destabilizing activity of Op18, which in turn promotes increased tubulin polymerization. Hence, the present study shows that Op18 has the potential to regulate the MT system in response to external signals such as cAMP-linked agonists.
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Introduction |
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MICROTUBULES (MTs)1 are important for many
cellular processes, for example in organelle
transport, organizing the cytoplasm, and for intracellular movement of cell surface receptors (for review
see Cole and Lippincott-Schwartz, 1995). During cell division, large arrays of MTs form the mitotic spindle, which
segregates condensed chromosomes (for review see Hyman and Karsenti, 1996
). The remarkable dynamic behavior of MTs, termed "dynamic instability," can be described
as stochastic transitions at MT ends between phases of
growth and shrinkage, with transitions from shrinking to
growing called "rescues," and the reverse called "catastrophes" (for review see Desai and Mitchison, 1997
).
The multitude of cellular functions involving MTs and
their dynamic behavior makes regulation of MT assembly
a central cell biological issue. Little is known, however,
about regulation of MT dynamics in response to external
and internal cellular signals. Most studies on this topic
have been concerned with microtubule-associated proteins (MAPs), a group of proteins that regulate MT dynamics by direct binding (for review see Mandelkow and
Mandelkow, 1995). Many reports have described how phosphorylation of MAPs by various protein kinases, including
the cAMP-dependent protein kinase (PKA), decreases
their MT-binding activities. However, the significance of
these findings for regulation of MT dynamics in intact cells remains to be established (for review see Maccioni and Cambiazo, 1995
).
Studies on intact cells indicate the presence of MT-regulatory factors that oppose the MT-stabilizing activity of
MAPs by promoting catastrophes and thereby increasing
the dynamics of MTs (for review see McNally, 1996). Two
such factors that increase the catastrophe rate have recently been identified, namely a kinesin-like protein,
XKCM1 (Walczak et al., 1996
) and a cytosolic protein, oncoprotein 18 (Op18; Belmont and Mitchison, 1996
). Op18
has been given many names in the literature (e.g., p19,
19K, metablastin, prosolin, and stathmin) due to its complex pattern of phosphorylation and its elevated expression in a variety of human malignancies (for review see
Belmont et al., 1996
). By using a conditional expression
system and kinase target site-deficient mutants of Op18,
we have previously demonstrated that Op18 is a phosphorylation-responsive regulator of MT dynamics in intact
cells; and furthermore, that multisite phosphorylation during mitosis downregulates the MT-destabilizing activity of
Op18 (Marklund et al., 1996
). These findings were extended in a subsequent study, where it was shown that mitosis-specific phosphoisomers of Op18 have severely reduced in vitro activities toward purified tubulin (Larsson
et al., 1997
). Taken together, the combined genetic, biochemical, and morphological data indicates that downregulation of Op18 activity is essential for formation of the
mitotic spindle.
Studies in brain tissue and various cell lines have shown
that Op18 is phosphorylated on four residues, namely Ser-16, Ser-25, Ser-38, and Ser-63 (Labdon et al., 1992; Beretta
et al., 1993
; Marklund et al., 1993a
; Wang et al., 1993
). All
four phosphorylation sites are subject to regulation by external signals during the cell cycle. During mitosis, Op18 is
phosphorylated on all four sites to high stoichiometry;
Ser-25 and Ser-38 are phosphorylated by members of the
cyclin-dependent kinase family, and Ser-16 and Ser-63 by
an as yet unidentified protein kinase (Brattsand et al., 1994
;
Larsson et al., 1995
). Op18 is also phosphorylated by several kinase systems in response to external signals. Thus, Op18 is phosphorylated on Ser-25 by members of the mitogen-activated protein (MAP) kinase family in response
to phorbol esters, T cell antigen receptor stimulation, and
nerve growth factor (Beretta et al., 1993
; Marklund et al.,
1993a
,b). Op18 is also phosphorylated by Ca2+/calmodulin-dependent kinase IV/Gr (CaMK IV/Gr) on Ser-16 in
response to calcium signals in specific cells that express this
kinase (Marklund et al., 1993a
; Melander Gradin et al., 1997).
Finally, cAMP-linked agonists have been shown to increase Op18 phosphorylation (Schubart et al., 1987
), and
subsequent studies have shown PKA-mediated in vitro
phosphorylation of Op18 on both Ser-16 and Ser-63 (Beretta et al., 1993
).
As outlined above, recent studies have shown that phosphorylation-mediated downregulation of Op18 activity is essential in human cell lines during formation of the mitotic spindle. These results imply that Op18 activity is of primary importance in regulating MT dynamics during the interphase of the cell cycle, and not during mitosis. It follows that kinase systems regulated by external signals, such as MAP kinase, PKA, and CaMK IV/Gr, may have important functions in regulating the MT-destabilizing activity of Op18. We have recently addressed this question by studying the consequences of CaMK IV/Gr-mediated phosphorylation of Op18. This study demonstrated that CaMK IV/ Gr-mediated phosphorylation of Ser-16 suppresses Op18 activity in intact cells (Melander Gradin et al., 1997). However, the data did not exclude regulatory importance of constitutive phosphorylation on additional sites of Op18. Importance of additional sites appears likely, because subsequent in vitro studies in our laboratory have suggested that phosphorylation on multiple sites of Op18 synergize in downregulation of Op18 activity. PKA has the potential to phosphorylate two specific sites of Op18 and may, therefore, be a very efficient regulator of Op18 activity and consequently also of the MT system. The present study addresses this possibility by evaluating the MT-regulatory role of all four potential phosphorylation sites of Op18 in response to PKA activity.
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Materials and Methods |
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DNA Constructs and Transfections
The coding region of a cDNA encoding a c-myc epitope-tagged C subunit of PKA (NcoI to ApaI fragment; Orellana and McKnight, 1992
), was
cloned into pMEP4 (Groger et al., 1989
) as a BamHI to HindIII fragment
after linking to the 5
untranslated region of Op18. The pMEP4-based
Op18 derivatives and
CaMK IV/Gr used in this study has been described
together with standard and co-transfection protocols for the K562 leukemia cell line (Marklund et al., 1994
; Larsson et al., 1995
; Melander Gradin
et al., 1997). The pMEP4 shuttle vector contains the Epstein-Barr virus
origin of replication and the EBNA-1 gene to allow high-copy episomal
replication, and the hph gene, which confers hygromycin B resistance in
mammalian cells (Groger et al., 1989
). Conditional expression of various Op18 derivatives was achieved by using the hMTIIa promoter, which can
be suppressed by low concentrations of EDTA (50 µM) and induced by
Cd2+ (0.03-0.2 µM; Marklund et al., 1994
). Transfected cells were cultured in a medium containing EDTA (50 µM) that has been specifically designed to support cell growth under conditions that minimize expression from the hMTIIa promoter (Marklund et al., 1994
). About 50-70%
of all pMEP4-transfected cells surviving electroporation were resistant to
hygromycin B (0.25 mg/ml; Boehringer Mannheim, Mannheim, Germany)
and mock-transfected cells were killed within 3 d. Experiments were routinely performed 5-6 d after transfection.
Analysis of MT Polymerization Status, Op18 Phosphoisomers, SDS-PAGE, and Western Blot
Preparation of total cellular proteins and separation of proteins by 10-
20% gradient SDS-PAGE has been described (Marklund et al., 1994). The
cellular content of MT polymers was determined by extracting soluble tubulin in an MT-stabilizing buffer followed by quantification of tubulin in
the particulate and soluble fraction as described (Minotti et al., 1991
; Marklund et al., 1996
). Affinity-purified anti-Op18, specific for the COOH-terminal (anti-Op18:34-149) or NH2-terminal (anti-Op18:2-33), was prepared
and used for Western blot analysis as described (Brattsand et al., 1993
).
For detection of epitope-tagged proteins, an anti-c-myc antibody (AB-1,
Oncogene Science Inc., Manhasset, NY) or the anti-Flag-M2 antibody
(Kodak) was used. 125I-protein A or the enhanced chemiluminescence detection system (Amersham Buchler GmbH, Braunschweig, Germany),
were used to reveal bound antibodies, as indicated. PhosphorImager analysis of radioactive bands was used for quantification. As a control for
equal loading, the relevant part of filters was routinely probed with rabbit anti-triose-phosphate isomerase (Brattsand et al., 1993
). To analyze Op18
phosphoisomers, we used a native PAGE system that separates Op18 according to the charge differences introduced by each of the four identified
phosphorylations (Marklund et al., 1993b
).
In Vitro Phosphorylation of Op18, Cross-linking of Op18-Tubulin Complexes and Determination of the MT-destabilizing Activity of Op18
Wild-type (wt) and kinase target site-deficient mutant derivatives of purified Escherichia coli-derived Op18 were prepared as described (Brattsand
et al., 1993). Purified Op18 was in vitro phosphorylated to high stoichiometry with PKA (New England BioLabs, Inc., Beverly, MA) by incubating
2.8 U of kinase/µg of Op18 in 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2 and
500 µM ATP for 3 h at 30°C. Reactions were terminated by heating to
75°C for 10 min and precipitated overnight at
20°C with 6 vol of MeOH
containing 1% sucrose. The precipitate was washed twice with 75% MeOH
containing 1% sucrose, dried under vacuum, and resuspended in PEM
buffer (80 mM Pipes, 1 mM EDTA, 1 mM Mg2+, pH 6.8). To remove
PKA that remained insoluble after MeOH precipitation, the final preparation of phosphorylated Op18 was clarified by centrifugation (15 min,
14,000 g). The concentration of the resulting phosphorylated Op18 was
calculated using SDS-PAGE comparison with a standard recombinant
Op18 preparation. As controls, in vitro phosphorylations were also performed either in the absence of ATP or by using mutants of Op18 lacking
the specific phosphorylation sites. In these cases, tubulin binding and MT-destabilizing activity of Op18 was unaffected. Therefore, the observed effects of Op18 phosphorylation could be attributed to addition of phosphate groups to specific Ser residues. Purified bovine tubulin was obtained from Cytoskeleton (Denver, Colorado). For cross-linking studies, Op18 (1 µM) and tubulin (10 µM) was incubated in 30 µl of PEM buffer
with 1 mM GTP. After 120 min on ice, 6 µl of glutaraldehyde (0.3%) was
added and the sample was incubated at 18°C. The reaction was quenched at
various times by addition of 1 vol 2-mercaptoethanol (10%)/glycine (0.2 M),
precipitated in 66% acetone, and the cross-linked Op18-tubulin complexes analyzed by SDS-PAGE. In vitro assembly of tubulin (4 µM) in the
presence of various amounts of Op18 was performed in assembly buffer
(25 µl of PEM buffer with 1 mM GTP, containing an additional 4 mM
Mg2+, 10% glycerol and 4 µg/ml taxol) as previously described (Larsson et al., 1997
). Determination of tubulin and Op18 protein mass, by analysis
of amino acid composition, was performed as described (Brattsand et al.,
1993
).
Immunofluorescence and Flow Cytometric Analysis
Cells were extracted with MT-stabilizing buffer (see above) containing
0.05% saponin and 10 µg/ml RNase. Cells were fixed in 4% paraformaldehyde and 0.5% glutaraldehyde for 15 min followed by quenching with
NaBH4, and thereafter stained with anti--tubulin (clone B-5-1-2, Sigma
Chemical Co., Poole, UK). Bound antibodies were revealed by fluorescein conjugated rabbit anti-mouse Ig and DNA was stained with 0.1 µg/
ml propidium iodide. Cells were mounted using 1 mg/ml of p-phenylendiamine in PBS with 80% glycerol and analyzed by epifluorescence. MT fluorescence was also analyzed by flow cytometry as described (Roos et al.,
1993
).
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Results |
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Regulation of MT Polymerization Status by Ectopic Expression of Op18, CaMK IV/Gr, and PKA
To investigate MT-regulatory properties of Op18 and two of
its cognate protein kinases, we expressed epitope-tagged
derivatives of Op18-wt, a constitutively active mutant of
CaMK IV/GR (CaMK IV/Gr), and the catalytic C
subunit of PKA in K562 leukemia cells using the Cd2+-inducible pMEP4 shuttle vector system. Western blot analysis of
lysates from transfected cells reveals induced expression
of the indicated gene products within 4 h of Cd2+ addition
(Fig. 1 A, note that the band migrating below endogenous Op18 is caused by proteolysis). Quantification of data from
several experiments shows that Cd2+ treatment results in
5-20-fold induction of Op18 and 30-100-fold induction of
CaMK IV/Gr and PKA.
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Previous studies have shown that CaMK IV/Gr phosphorylates Op18 on a single site, namely Ser-16, both in
vitro and in intact cells. Previous in vitro experiments have
shown that PKA phosphorylates Op18 on two sites, namely
Ser-16 and Ser-63. To analyze Op18 phosphoisomer composition in cells induced to express either CaMK IV/Gr
or PKA, we used a native gel system (Marklund et al.,
1993b
). The data shows, in agreement with a previous report (Melander Gradin et al., 1997), that essentially all endogenous Op18 molecules are phosphorylated on a single
site in cells induced to express
CaMK IV/Gr (Fig. 1 B).
In agreement with the site preference of PKA in vitro (Beretta et al., 1993
), it is also shown that overexpression of
this kinase results in phosphorylation of two sites of Op18.
We have recently shown that overexpression of Op18
causes destabilization of interphase MTs and that this activity of Op18 can be suppressed by phosphorylation (Marklund et al., 1996; Melander Gradin et al., 1997). The response to ectopic Op18 expression, or two of its cognate
kinases, on the level of cellular content of MT polymers is
shown in Fig. 1 C. In agreement with our previous reports,
ectopic expression of Op18 results in depolymerization of
MTs, whereas expression of
CaMK IV/Gr results in increased MT polymers. Most importantly, it is also shown
that expression of PKA increased cellular content of MT
polymers with even higher efficiency than
CaMK IV/Gr.
Induced expression of CaMK IV/Gr has been shown
to result in a partial (~50%) degradation of endogenous
Op18. Both the phosphorylation of Op18 on Ser-16 and
the associated partial degradation of Op18 are likely to
contribute to the observed increase of cellular content of
MT polymers in response to
CaMK IV/Gr expression
(Melander Gradin et al., 1997). Fig. 2 shows the time course
of Cd2+-induced PKA expression and the consequences of
induced expression on endogenous Op18 protein levels
and cellular content of MT polymers. The data reveal that
similarly to
CaMK IV/Gr, PKA-mediated phosphorylation induces a partial degradation of Op18. Moreover, it is
also evident that the cellular content of MT polymers increases in parallel with Op18 degradation. Cd2+ induces a
10-fold increase of PKA expression within 2 h. At this early time point, both downregulation of Op18 and increased MT polymers are readily observed.
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PKA-mediated Phosphorylation Switches Off the MT-destabilizing Activity of Op18
To dissect the function of PKA-specific Op18 phosphorylation at distinct sites, we used a previously described co-transfection protocol (Melander Gradin et al., 1997). This protocol allows regulated expression of two co-transfected gene products and efficient phosphorylation of ectopic Op18 by a cognate protein kinase. It is shown by Western blot analysis in Fig. 3 A that PKA can be efficiently co- expressed with either Op18-wt or Op18-S16,63A. In agreement with the data in Fig. 1 A, levels of endogenous Op18 are decreased after induced expression of PKA alone (note that phosphorylation results in a minor shift in migration). However, in contrast to the endogenous Op18, we do not detect a significant decrease in the levels of ectopic Op18 protein because of rapid-induced expression during the time course of the experiment. Hence, the present co-transfection protocol is useful to explore the effect of specific regulatory phosphorylations of Op18 on the MT-regulatory activity of Op18, with minimal interference on the level of protein degradation.
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To determine if PKA phosphorylation regulates Op18 activity, we analyzed alterations in cellular content of MT polymers in co-transfected cells. As expected, Cd2+ treatment of cells transfected with vector-Co alone was without effect, whereas co-transfection of vector-Co with either Op18-wt or Op18-S16,63A results in Cd2+-induced depolymerization of MTs (Fig. 3 B). Moreover, as predicted from the data shown above, co-transfection of vector-Co and PKA results in a Cd2+-induced increase in MT polymers. Most significantly, co-transfected PKA completely suppresses the MT-depolymerizing activity of Op18-wt, but not of the Op18-S16,63A PKA target site-deficient mutant.
The simplest interpretation of the results outlined above is that PKA-mediated phosphorylation of Ser-16/Ser-63 switches off the MT-regulatory function(s) of Op18. However, the overexpressed and PKA phosphorylated Op18 protein may have some aberrant MT-regulatory properties that are not detected by analysis of total cellular content of MT polymers. Therefore, we analyzed the appearance of MTs in co-transfected cells by immunofluorescence. The analysis was performed on cells extracted with an MT-stabilizing buffer before fixation, and nuclear DNA was stained with propidium iodide. The immunofluorescence shown in Fig. 4 A demonstrates a characteristic interphase network of MTs in cells induced to express PKA either alone or together with Op18-wt. Moreover, cells induced to express any of the two Op18 derivatives without PKA, or Op18-S16,63A together with PKA, contained very few MTs. The spherical shape of the K562 leukemia cells used in this study is not optimal for morphological studies of MTs and it is not possible to analyze the length or number of individual MTs. Nevertheless, detailed examination of cells using high magnifications failed to reveal any morphological differences in the MT network displayed in cells expressing PKA alone or together with Op18-wt.
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To quantify MT-specific immunofluorescence and to analyze heterogeneity within the cell population, we used flow cytometry. The resulting histograms show that expression of PKA alone results in a 1.8-fold homogeneous increase in mean fluorescence of the major cell population (Fig. 4 B, note log scale). It is also shown that induced expression of Op18 results in an almost 10-fold decrease in MT specific fluorescence. Most significantly, PKA restores the fluorescence in cells co-transfected with Op18-wt, but not Op18-S16,63A.
Taken together, the result in Fig. 4 suggests that PKA expression induces a homogeneous increase of cellular content of MT polymers without gross alterations in MT morphology. Moreover, cells overexpressing Op18 in its PKA phosphorylated form show no detectable phenotype on the level of MT morphology. These results are compatible with a simple model where PKA promotes increased tubulin polymerization by phosphorylation of Op18, which switches off the MT-destabilizing activity of Op18.
The Role of Individual Phosphorylation Sites for PKA-mediated Modulation of Op18 Activity
Previous studies have shown that both Ser-16 and Ser-63
are phosphorylated by PKA in vitro. This is in line with
the appearance of di-phosphorylated endogenous Op18 in
transfected cells induced to express a catalytic subunit of
PKA (Fig. 1 B). An earlier study has shown that overexpression of Op18 for 24 h results in hyper-phosphorylation of Op18, mostly on Ser-38 (Marklund et al., 1996). This
phenomenon is most likely caused by Op18-induced MT
depolymerization, because it has been shown that drug-
induced depolymerization results in activation of a MAP
kinase-related kinase (Shinohara-Gotoh et al., 1991
). In
the present study, we induced expression for a short time
period (4-5 h). This results in a more modest phosphorylation level of the overexpressed Op18-wt protein, as compared to 24 h of induced expression, with ~20% of all
Op18 phosphorylated on a single site (Fig. 5). However, by
inducing expression of Op18-wt in the presence of PKA,
~85% of all cellular Op18 is phosphorylated on one or
more sites (Fig. 5). Hence, unlike the situation of endogenous Op18 in cells induced to express PKA alone (Fig. 1), it is evident that not all ectopic Op18 molecules are phosphorylated on two sites in cells that also overexpress PKA.
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To analyze the site-specificity of PKA-mediated phosphorylation of Op18, we determined the phosphorylation
status of a panel of phosphorylation site-deficient Op18
mutants co-expressed with either vector-Co or PKA (Fig.
5). Op18 can be phosphorylated on four specific sites in
K562 cells (Larsson et al., 1995). The Op18-S16,25,38,63A
derivative has all of these four sites mutated and the expressed protein is, as expected, essentially unphosphorylated if expressed together with PKA. It should be noted
that Op18 derivatives without epitope-tag were expressed
in this experiment, and the low level of phosphoisomers
observed for Op18-S16,25,38,63A is due to the endogenous gene product. From the analysis shown in Fig. 5, it is
evident that Ser-63 is the predominant site phosphorylated by PKA, because almost 75% of the Op18-S16,25,38A mutant protein is phosphorylated on a single site. In contrast,
only ~30% of the Op18-S25,38,63A mutant protein is
phosphorylated on a single site.
To dissect the functional role of specific phosphorylation sites during PKA-mediated suppression of Op18 activity, we determined cellular content of MT polymers in cells co-transfected with PKA and the panel of kinase target site-deficient mutants of Op18 presented in Fig. 5. The results in Fig. 6 show that phosphorylation of Ser-16/Ser-63 is both sufficient and necessary for efficient PKA-mediated suppression of Op18 activity (compare Op18-wt and Op18-S25,38A). Single-site phosphorylation on Ser-63 only results in a partial suppression of Op18 activity and single-site phosphorylation of Ser-16 has even less effect (compare Op18-S25,38,63A with Op18-S16,25,38A). Finally, and as expected, PKA has no effect on the MT-destabilizing activity of the Op18-S16,25,38,63A mutant protein.
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In interpreting the result shown in Fig. 6, the stoichiometry of phosphorylation shown in Fig. 5 should be considered. Thus, only ~30% of the overexpressed Op18-S25, 38,63A protein is phosphorylated on its remaining site, Ser-16. Nevertheless, the results suggest that dual phosphorylation on Ser-16 and Ser-63 is of importance for efficient PKA-mediated suppression of Op18 activity in intact cells.
Kinetics of Dual Phosphorylation by PKA
PKA phosphorylates both Ser-16 and Ser-63 of Op18 in intact cells, and both mono- and di-phosphorylated Op18 are generated (Fig. 5). To evaluate potential cooperativity during PKA-mediated phosphorylation of Ser-16 and Ser-63, the time course for generation of multi-phosphorylated Op18 species in the presence of purified PKA was determined (Fig. 7 A). The data reveals transient accumulation of mono-phosphorylated Op18 and subsequent accumulation of di-phosphorylated Op18, until a time point where Op18 is predominantly phosphorylated on two sites. Only low levels of tri-phosphorylated Op18 are generated, which indicates that Ser-16 and Ser-63 are the sole physiological PKA target sites of Op18. Because >50% of the available Op18 substrate is mono-phosphorylated at early time points, before significant amounts of di-phosphorylated Op18 are present, the data shows that Ser-16 and Ser-63 are not simultaneously phosphorylated by PKA.
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To further address if Ser-16 and Ser-63 of Op18 are independently phosphorylated by PKA, the rate of in vitro phosphorylation of the Op18-S63A and Op18-S16A mutants was compared to that of Op18-wt. The results show that Ser-63 is phosphorylated three- to fourfold faster than Ser-16 (Fig. 7 B). Because the sum of the rates of phosphorylation on each site (21 + 73 = 94 pmol PO4/min) is close to the phosphorylation rate observed using the wild-type Op18 substrate (96 pmol PO4/min), the results suggest that Ser-16 and Ser-63 are independently phosphorylated by PKA. This, together with the site preference of PKA in vitro, is in line with the distribution of Op18 phosphoisomers observed in cells co-transfected with PKA and a panel of kinase target site-deficient mutants (Fig. 5). Finally, native gel analysis shows that extensive PKA-mediated phosphorylation of an Op18-wt substrate, or the indicated mutated substrates, results in essentially homogeneous preparations of specific Op18 phosphoisomers (Fig. 7 B).
Dissection of the Regulatory Role of Ser-16 and Ser-63 Phosphorylation for Op18 Activities towards Purified Tubulin
Op18 has been shown to bind to tubulin, an interaction
that is likely to be of functional importance (Belmont and
Mitchison, 1996). To analyze the effect of Op18 phosphorylation on tubulin binding, we used glutaraldehyde to preserve Op18-tubulin complexes. As shown by SDS-PAGE
separation in Fig. 8 A, addition of glutaraldehyde preserves an 83-kD complex that is recognized by antibodies
against Op18. As expected, this complex is also recognized
by anti-
-tubulin (data not shown). We have previously
reported that cross-linking with the zero-length cross-linker 1-ethyl-3(3-[dimethylamino]propyl)carbodiimide, results in two distinct Op18-tubulin complexes migrating at
71 and 83 kD (Larsson et al., 1997
). As shown in Fig. 8,
cross-linking with glutaraldehyde reproduces the 83-kD
but not the 71-kD complex. However, both protocols seem
to generate predominantly Op18 cross-linked to
-tubulin,
because the complexes react poorly with two different anti-
-tubulin antibodies (Larsson et al., 1997
; and data
not shown). It should be noted that cross-linking of Op18
to tubulin seems to be a very specific process, as nonrelated proteins do not interfere with the process, and Op18
is not cross-linked to tubulin that has been made polymerization incompetent by 5 min at 60°C (data not shown).
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For functional dissection of individual phosphorylation
sites, Op18-S63A, Op18-S16A, and Op18-wt substrates
were phosphorylated to essentially complete stoichiometry by PKA (see native gel shown in Fig. 7 B). As shown in
Fig. 8 A, single-site phosphorylation on either Ser-16 or
Ser-63 results in a partial reduction of the Op18-tubulin
complex (~50%) as compared to the nonphosphorylated Op18 protein. Moreover, and in agreement with our previous study (Larsson et al., 1997), phosphorylation on both
sites essentially abolished complex formation. Similar effects of mono- and diphosphorylation of Op18 on Ser-16/
Ser-63 were observed by using the 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide cross-linker (data not shown).
Hence, single-site phosphorylation on either Ser-16 or Ser-63 have significant functional consequences on Op18-
tubulin complex formation but the effect of di-phosphorylation is clearly synergistic.
We also determined functional consequences of Ser-16 and Ser-63 phosphorylation of Op18 during MT polymerization. The assay was performed in the presence of taxol and the activity of Op18 is, therefore, likely to reflect inhibition of polymerization rather than destabilization of MTs. As shown in Fig. 8 B, unphosphorylated Op18 completely inhibits MT polymerization at ~4 µM concentration, whereas dual phosphorylation on Ser-16 and Ser-63 neutralizes the inhibitory activity of Op18. The effect of single-site phosphorylation of either Ser-16 or Ser-63 alone was small and barely detectable in this assay system. Taken together, dual phosphorylation on Ser-16 and Ser-63 blocks both tubulin complex formation and the inhibitory activity during MT polymerization, whereas the effect of single-site phosphorylation could only be detected with certainty by analyzing tubulin complex formation.
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Discussion |
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By using a conditional expression strategy, we show that the catalytic subunit of PKA mediates a rapid and dramatic increase in cellular MT-polymer content in parallel with dual phosphorylation and partial degradation of Op18. PKA is a multi-functional kinase with a multitude of cytosolic and nuclear protein substrates. To evaluate a potential role of Op18 phosphorylation for PKA regulation of the MT system, a panel of kinase-target-deficient mutants of Op18 were co-transfected with PKA. The result demonstrated that PKA switches off the MT-destabilizing activity of Op18 in intact cells by dual phosphorylation of Ser-16/Ser-63. In vitro studies confirmed the importance of dual phosphorylation of these two sites in switching off Op18 activity. Hence, the model system used in this study demonstrated the potential of PKA to regulate the MT system via Op18 phosphorylation.
Immunofluorescence analyses suggest that PKA expression induces a homogeneous increase of cellular content of MT polymers without gross alterations in MT morphology. Moreover, cells overexpressing Op18-wt together with PKA show no detectable phenotype on the level of MT morphology (Fig. 4). Hence, it appears that Op18 in its PKA-phosphorylated form is devoid of all of its MT-regulatory properties. These results are compatible with a simple model where PKA promotes increased tubulin polymerization by phosphorylation of Op18, which in turn directly switches off the MT-destabilizing activity of Op18.
This study demonstrates that dual phosphorylation of
Ser-16 and Ser-63 inactivates both tubulin-binding activity
and MT polymerization inhibitory activity of Op18. This
may appear contradictory to a recent study (Horwitz et al.,
1997), in which the potential mimic of phosphorylation
provided by Asp substitutions at phosphorylated sites was
analyzed. In this study the authors concluded that specific Asp substitutions decreased the MT-destabilizing activity
of a panel of glutathione S-transferase-Op18 fusion protein derivatives as analyzed by micro-injection of a cell line.
However, in vitro assays failed to reveal any effect by Asp
substitutions on the MT polymerization inhibitory activity
of the Op18 fusion proteins. To explain these results, the
authors proposed that cellular factors are required to reveal the inhibitory effect of Op18 phosphorylations. Because our studies show that phosphorylation on specific
sites of the native Op18 protein clearly inhibits its in vitro
activity, it appears that Asp substitutions at phosphorylation sites of a fusion protein only partially mimic the regulatory effects of phosphorylation.
As outlined in the introduction section, Op18 has been
independently identified by several groups. In at least
three cases, Op18 was initially identified as a major phosphorylation event in response to increased cAMP levels,
induced by either specific hormones or drugs (Brattin and
Portanova, 1981; Schubart, 1982; Sobel and Tashjian, 1983
).
This together with in vitro phosphorylation studies, and
site-specific phosphorylation of Op18 in response to ectopic PKA expression shown herein, suggest that Op18 is a physiological substrate for PKA. However, Op18 is not
phosphorylated in response to PKA activation in all cell
types investigated. Hence, published results suggest that,
whereas Op18 is clearly a likely major cytosolic substrate
for PKA in cell types of neural origin, Op18 phosphorylation is not significantly increased in response to cAMP-linked agonists in hematopoietic/lymphoid cell types (Mary
et al., 1989
; Strahler et al., 1992
). The reason for these differences is not clear, but it seems possible that PKA activity is less abundant in some cell types, and/or that most of
the catalytic subunits are translocated to the nucleus upon activation. Nevertheless, Op18 is clearly efficiently phosphorylated in response to cAMP signaling in many cell
types, which the present study predicts has implications for
regulation of the MT system.
PKA has previously been implicated in the regulation of
the MT systems. For example, the two neuronal MAPs,
MAP2 and tau, are phosphorylated on the same sites in intact cells in response to cAMP-linked agonists as they are
by PKA in vitro (Jefferson and Schulman, 1991; Fleming
and Johnson, 1995
). It is generally believed that different
signal transduction cascades may control MT dynamics by
phosphorylation of MAPs. In functional studies, phosphorylation has been shown to inhibit to various extents the MT-stabilizing in vitro activity of MAPs (Jameson and Caplow, 1981
; Lindwall and Cole, 1984
; Drechsel et al., 1992
;
Hoshi et al., 1992
; Masson and Kreis, 1995
; Ookata et al.,
1995
; Trinczek et al., 1995
). Hence, from these studies it
can be predicted that phosphorylation of MAPs in intact
cells should result in clearly visible disruption of MTs. Although many studies have been concerned with phosphorylation of MAPs by PKA, there is to our knowledge no
study demonstrating PKA-mediated disruption of MTs in
intact cells.
If, as outlined above, PKA modulates the MT system by
phosphorylation of MAPs, previous studies predict that
activation of the kinase should destabilize MTs. Using a
conditional overexpression strategy, the present study shows
the opposite result, namely, that PKA activity mediates an
increase in cellular MT polymer content. Moreover, PKA
can efficiently switch off the potent MT-destabilizing activity of Op18 in intact cells, provided that the Ser-16 and Ser-63 Op18 target sites are intact, suggesting an Op18-
dependent mechanism. Another indication that PKA regulation of MTs is primarily mediated by Op18 in K562
cells comes from the observation that specific Op18 mutants can completely abolish the MT-stabilizing effect of
PKA. If PKA activation would mediate increased MT
polymers primarily by activating MT-stabilizing proteins
(e.g., MAPs), it would be anticipated that the resulting
MTs would show increased stability, and therefore be partially resistant to the destabilizing activity of specific mutants of Op18. Co-transfection experiments show that this
is not the case because PKA expression is without any effect on the MT system in the presence of the Op18-mutants with Ser-16 and Ser-63 to Ala substitutions (Figs. 3
and 6). However, although Op18 appears to be a major
regulator of the MT system in the K562 erythroleukemia
cell line, this does not need to be the case in all tissues/cell
lines. It is also possible that other phosphorylation responsive proteins, functionally related to Op18, are involved in
regulation of the interphase MT network. If this is the
case, redundancy may explain why mice with both alleles of the Op18 gene disrupted by homologous recombination
survive and show normal development (Schubart et al.,
1996).
The MT system seems, in general, less intimately associated with second messenger systems than is the actin system, and little is known about its regulation in response to
external signals (Mitchison, 1992). However, the present
and a previous study by us show that the recently identified MT-regulating protein, Op18, links the MT system and
at least two second messengers, namely cAMP and Ca2+.
In our previous study, a constitutively active mutant of
CaMK IV/Gr was shown to suppress the MT-destabilizing
activity of Op18-wt by phosphorylating Ser-16 (Melander
Gradin et al., 1997). However, the importance of constitutive phosphorylations on additional sites for suppression
of Op18 activity was not addressed. Thus, overexpressed
Op18 shows significant levels of constitutive phosphorylation on Ser-38, which is likely to have a synergistic effect
together with CaMK IV/Gr-mediated Ser-16 phosphorylation (Larsson et al., 1997
). In the present study, the MT-regulatory role of all four potential phosphorylation sites
of Op18 was determined in response to induced expression of PKA. The result shows that PKA phosphorylates both Ser-16 and Ser-63 in intact cells, with preference for
the latter site. Moreover, it was also demonstrated that
PKA-mediated dual phosphorylation of Op18 on these
two Ser residues is both necessary and sufficient for efficient regulation of the MT system. As predicted from the
dual specificity of PKA, comparison in the same experiment reveals that PKA is more potent than CaMK IV/Gr in inducing increase in cellular content of MT polymers
(Fig. 1). Furthermore, the importance of dual phosphorylation of Op18 was also evident in co-transfection experiments in which Op18 was overexpressed. Hence, although
CaMK IV/Gr mediates complete phosphorylation on Ser-16
of overexpressed Op18, the resulting MT-destabilizing activity of Op18-wt was not as efficiently suppressed as observed in this study by co-transfection of PKA (Fig. 3). This is despite the observation that ectopic PKA did not
mediate complete phosphorylation of its two target sites of
Op18, as indicated by quantification of Op18 phosphoisomers (Fig 5). Because the kinase activity of ectopic PKA
was not sufficient to dual phosphorylate overexpressed
Op18 to completion, the data does not provide any information on the relative MT-regulatory importance of Ser-16 versus Ser-63 in intact cells (Figs. 5 and 6). However, in
vitro experiments suggested similar effects of phosphorylation of either of the two Ser residues and a clear-cut synergistic effect of phosphorylating both sites (Fig. 8).
The effects of phosphatase/kinase inhibitors on MT dynamic instability was recently investigated in living newt
lung epithelial cells (Howell et al., 1997). Significant and
rapid (within 1 min) alterations of the dynamic behavior of
individual MTs was observed in response to all inhibitors
tested. The authors proposed that both MAPs and Op18
are likely mediators of the MT-regulatory response to drug-induced alterations of protein phosphorylation. Moreover, a previous report has also shown that phorbol ester treatment of macrophages results in a rapid (detectable within
minutes) increase in MT length and number (Robinson
and Vandre, 1995
). Phorbol esters are specific activators
of protein kinase C, which is an upstream activator of the
MAP kinase in leukocytes (Cantrell, 1994
). Because Op18
is a major cytosolic substrate of the MAP kinase (Marklund
et al., 1993b
), it seems possible that the reported effect is
at least in part due to phosphorylation-mediated inactivation of Op18. In addition, studies on murine embryological
fibroblasts have shown that stimulation with thrombin,
epidermal growth factor, and phorbol esters increases the
cellular content of MT polymers (Ball et al., 1992
). Because Op18 is likely to be phosphorylated in response to
all of these three mitogens, it seems possible also in these
cases that inactivation of Op18 contributes to increased MT polymers.
One potential mechanism for increased cellular content
of MT polymers in response to external signals may be
stimulation of tubulin synthesis. In the studies on macrophages and fibroblasts, mentioned above, changes in the
total pool of tubulin were not quantified. However, because both studies reported significant responses within an
hour after stimulation, it seems unlikely that an increase in
the total pool of tubulin is responsible for the observed increase of cellular MT polymers. In the present study, alterations of MT polymers in response to overexpressed PKA,
which was detected within 2 h (Fig. 2), was determined by quantification of both polymerized and soluble tubulin.
Over a time course of 12 h we did not observe any detectable alteration in the total pool of tubulin by overexpressing PKA or Op18 (data not shown). Hence, in the present
study, and most likely in the two studies mentioned above,
external signals appear to induce an early increase in MT
polymers in the absence of alterations of the tubulin pool
size. In a longer time perspective, however, the rate of tubulin synthesis is most likely changed because the level of
tubulin heterodimers is regulated by an autoregulatory mechanism, that is related to the pool size of unpolymerized tubulin heterodimers (Cleveland et al., 1981; Pachter
et al., 1987
).
![]() |
Footnotes |
---|
Received for publication 9 September 1997 and in revised form 5 November 1997.
Address all correspondence to Martin Gullberg, Department for Cell and Molecular Biology, University of Umeå, S-901 87 Umeå, Sweden. Tel.: (46) 90 7852532. Fax: (46) 90 771 420. E-mail: Martin.Gullberg{at}cmb.umu.seWe thank Dr. Victoria Shingler for critical reading of the manuscript, and
Dr. S. McKnight for providing a cDNA encoding the C subunit of the
cAMP-dependent protein kinase.
This work was supported by Swedish Natural Science Research Council, Lion's Cancer Research Foundation, University of Umeå (LP 797/91), the Foundation for Medical Research at the University of Umeå, and the Swedish Society for Medical Research.
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Abbreviations used in this paper |
---|
MAP, mitogen-activated protein; MAPs, microtubule-associated proteins; MT, microtubule; Op18, oncoprotein 18; PKA, cAMP-dependent protein kinase; wt, wild-type.
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