Biochimica et Biophysica Acta 1764 (2006) 299 – 306

http://www.elsevier.com/locate/bba

Casein kinases phosphorylate multiple residues spanning

the entire hnRNP K length
Michał Mikula b , Jakub Karczmarski a , Artur Dzwonek b , Tymon Rubel c , Ewa Hennig a,b ,
Michał Dadlez d,e , Janusz M. Bujnicki f , Karol Bomsztyk g , Jerzy Ostrowski a,b,⁎
a
Department of Gastroenterology, Medical Center for Postgraduate Education, Maria Słodowska-Curie Memorial Cancer Center, ul. Roentgena 5,
02-781 Warsaw, Poland
b
Institute of Oncology, 02-781 Warsaw, Poland
c
Institute of Radioelectronics, Warsaw University of Technology, Poland
d
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warszawa, Poland
e
Department of Biology, Warsaw University, Poland
f
Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology, Warsaw, Poland
g
Department of Medicine, UW Medicine at Lake Union, University of Washington, Seattle, WA 98109, USA
Received 14 September 2005; received in revised form 1 December 2005; accepted 4 December 2005
Available online 6 January 2006

Abstract

Heterogeneous Nuclear Ribonucleoprotein K (hnRNP K) is an RNA/DNA-binding protein involved in many processes that regulate gene
expression. K protein's pleiotropic action reflects the diversity of its molecular interactions. Many of these interactions have been shown to be
regulated by phosphorylation. K protein contains more than seventy potential phosphorylation sites. We used an integrated approach of mass
spectrometry and computer analysis to explore patterns of K protein phosphorylation. We found that in vitro a single kinase can phosphorylate K
protein on multiple sites spanning the entire length of the protein, including residues contained within the RNA/DNA-binding domains. 2-D gel
electrophoresis of K protein purified from cells identified 5–8 spots. Mass spectrometry of K protein isolated from proliferating cells and from
cells under oxidative stress revealed the same pattern of phosphopeptides. The structural implications of phosphorylation are discussed.
© 2005 Elsevier B.V. All rights reserved.

Keywords: hnRNPK; Immunoprecipitation; Casein kinases; Phosphorylation; Protein modeling; Mass spectrometry

1. Introduction the diversity of its molecular interactions which, in addition to

HnRNP K is an ancient RNA/DNA-binding protein that acts
in several subcellular compartments including the nucleus,
cytosol and mitochondria [1]. Along with other factors such as
YB-1 [2] and Sam68 [3], K protein is defining a novel class of
nucleic acid-binding factors involved in many of the processes
that compose expression of a large repertoire of genes [1]. The
involvement of K protein in a broad range of processes reflects
⁎ Corresponding author. Department of Gastroenterology, Medical Center for
Postgraduate Education, Maria Słodowska-Curie Memorial Cancer Center, ul.
Roentgena 5, 02-781 Warsaw, Poland. Tel.: +48 22 6440102; fax: +48 22
6447601.
E-mail address: jostrow@warman.com.pl (J. Ostrowski).
1570-9639/$ - see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbapap.2005.12.004
nucleic acids, includes multiple kinases, chromatin, transcrip-
tion, RNA processing and translation factors. Considering the
diversity of its interactions, the number of known K protein
domains that recruit these factors is rather small. In addition to
the three KH domains that bind RNA/DNA, there is central
unstructured KI region that mediates many of the known protein
interactions and the nuclear shuttling domain (Fig. 1)
K protein is phosphorylated on multiple residues and the level
of its phosphorylation is modulated by a host of stimuli including
IL-1 and insulin [4–6] as well as by oxidative stress [7].
Phosphoamino acid analysis [8] and Western blotting [4,7]
revealed that K protein is phosphorylated on serine, threonine and
tyrosine residues. Phosphorylation of K protein regulates its
interaction with RNA/DNA targets [4,9] and protein partners [7,10].
300 M. Mikula et al. / Biochimica et Biophysica Acta 1764 (2006) 299–306
Fig. 1. Schematic diagram of K protein structure and potential phosphorylation
sites. Domain composition of K protein is shown. NLS, nuclear localization
signal; KNS, nuclear shuttling domain; KH, K homology domain; RGG,
arginine–glycine–glycine boxes; SH3-BD, SH3-binding domains. There are 32
serines, 24 threonines and 17 tyrosines, together, 73 potential phosphorylation
sites.
K protein has 32 serines, 24 threonines and 17 tyrosines,
together 73 potential phosphorylation sites (Fig. 1), and among
those, eleven residues are known to be phosphorylated either in
vivo or in vitro [1]. The maximum number of phosphorylation
states can be calculated by the power formula 2n, where n is the
number of phosphorylated residues [4]. If the eleven known K
protein sites are the only residues that are phosphorylated, then
K protein could exist in more than two thousand states. But if
there are additional sites, say nine more for a total of twenty, K
protein could theoretically exist in more than a million states.
Mammalian cells have several hundred kinases [11], so there is
a sufficient number of enzymes that each site could be a target
of a single kinase. The high theoretical number of K protein
structural states could explain the diversity of its interactions.
2-D gel electrophoresis of phosphorylated K protein revealed
several discreet spots rather than a smear [12]. This observation
suggests a different model to that based on the power formula,
where a single kinase(s) could phosphorylate K protein on
multiple sites generating a much smaller number of covalent
states. To determine patterns of K protein phosphorylation we
used an integrated approach of MS and computer analysis.
2. Materials and methods
2.1. Cells
Rat hepatoma cells, HTC-IR, were grown in plastic cell culture flasks in
DME media supplemented with 10% FBS, 2 mM glutamine, penicillin (100
units/ml), streptomycin (0.01%), and humidified with 7/93% C02/air gas
mixture.
2.2. Purification of K protein
HTC-IR cells were sonicated three times for 3 s on ice (model 130-Watt
Cole-Parmer Ultrasonic Processor, set at amplitude 80) with 1.0 ml of
immunoprecipitation (IP) buffer (150 mM NaCl, 5 mM EDTA, 1% Triton X-
100, 0.5% NP-40, 50 mM Tris–HCl, pH = 7.5) containing the following
inhibitors: 10 μg/ml leupeptin, 0.5 mM PMSF, 0.5 mM dithiothreitol, 30 mM p-
nitrophenyl phosphate, 10 mM NaF, 0.1 mM Na3VO4, 0.1 mM Na2MoO4 and
10 mM β- glycerophosphate and kept on ice for 15 min. Cell lysates were
centrifuged (15.000 g, 4 °C, 20 min), supernatants were diluted in 10 ml IP
buffer and run by gravity through 4 columns: 3 containing 1 ml resin made up of
sepharose conjugated with rabbit IgG and one column containing 1 ml sepharose
conjugated with #54 antibody. Anti-K protein antibody #54, directed against the
C-terminus, or rabbit IgG were immobilized to Protein A Sepharose using
Disuccinimidyl Suberate (DDS) and Seize X Protein A immunoprecipitation Kit
(Pierce). After running the cellular extracts, columns were washed separately
with IP buffer containing 200 mM NaCl. Proteins were eluted from the resin,
first with IP buffer containing an appropriate salt concentration (0.6 or 1.0 M
NaCl), followed by 0.1 M glycine, pH = 2.6.
To generate recombinant His-K, cDNA of K protein was subcloned into
pET-28(+) expression vector (Novagen), and the plasmid was transformed into
E. coli BL21 DE3 pLysS cells (Novagen). Bacterial cells were harvested by
centrifugation, and following freezing and thawing, the bacterial pellet was
suspended in PBS containing 5 mM DTT, 0.1 mM leupeptin, 0.5 mM PMSF, 0.1
mM lysozyme and sonicated on ice. After centrifugation, recombinant His-K
protein was attached to His-binding resin (Novagen) in binding buffer
containing 50 mM NaH2PO4, pH = 8.0, 300 mM NaCl, 10 mM imidazole,
10% glycerol, 20 mM β−mercaptoethanol, 2 mM ATP, and 10 mM MgCl2.
Then, the resin was washed extensively with binding buffer containing 2 M
NaCl.
2.3. Phosphorylation in vitro
Purified bacterially expressed K protein was phosphorylated in vitro by
either casein kinase-1 (CK1) (Casein Kinase 1δ, rat, recombinant) or casein
kinase-2 (CK2) (Casein Kinase II purified from rat liver) in buffer containing 25
mM Tris, pH = 7.5, 200 mM NaCl, 0.1 mM ATP, 10 mM MgCl2 for 20 min at 30
°C. Phosphorylation of K protein with baculovirus-expressed Lck was
performed in the buffer containing 50 mM Tris, pH = 7.5, 0.1% Triton X-100,
0.1 mM ATP, 1 mM DTT, 10 mM MnCl2 for 20 min at 30 °C. Proteins were
digested, and the resulting phosphopeptides were analyzed by mass
spectrometry.
2.4. Mass spectrometry
Protein samples were analyzed by liquid chromatography-electrospray
mass spectrometry with collisional fragmentation (LC-ESI-MS-MS/MS). Prior
to analysis the proteins were reduced, alkylated (when necessary) and digested
with either trypsin (sequencing grade—Promega), V8 protease or cyanogen
bromide following a standard protocol. Eluted peptide mixture was applied to
RP-18 precolumn (LC Packings) using water containing 0,1% TFA as mobile
phase and than transferred to nano-HPLC RP-18 column (LC Packings, 75 μM
i.d.) using an acetonitrile gradient (0–50% AcN in 30 min) in the presence of
0.05% formic acid with the flow rate of 200 nl/min. Column outlet was directly
coupled to nano-Z-spray ion source of Q-Tof electrospray mass spectrometer
working in the regime of data dependent MS to MS/MS switch, allowing for 3
s sequencing scan for each detected peptide. A blank run ensuring lack of cross
contamination from previous samples preceded each analysis. The output list
of parent and daughter ions was used to search a database using MASCOT
(MatrixScience). Peptide identification and the presence of their covalent
modifications were verified by inspection of parent mass fragmentation
patterns using programs MassLynx and ProteinProspector.
2.5. Protein modeling
Prediction of K protein structure was carried out via the GeneSilico
metaserver, using a consensus of numerous methods [13]. The three-
dimensional model of protein K was built using the FRankenstein's Monster
method [14], with globular domains based on the coordinates of the KH3
domain NMR structure [15] and the rest of the protein modeled as a flexible coil.
Regions of protein disorder (flexible, without regular secondary structure) were
predicted using DISOPRED [16]. Buried residues and secondary structures were
identified based on the model using the DSSP method [17].
2.6. Prediction of phosphorylation sites
There are several bioinformatic tools currently available for identification of
phosphorylation sites in proteins [18,19]. Due to the high degree of variability in
consensus patterns around phosphorylation sites caused by the diversity and the
large number of kinases, and the still relatively small number of non-redundant,
experimentally verified sites, high-accuracy prediction of phosphorylation sites
remains an open area of research. The number of phosphorylation sites specific
for particular kinases is also relatively small. Therefore, most of computational
 M. Mikula et al. / Biochimica et Biophysica Acta 1764 (2006) 299–306 301

predictors do not discriminate between different enzymes and only predict
whether or not a given residue can be phosphorylated. Based on these
considerations, we decided to predict the putative phosphorylation sites in
protein K using three different methods for general phosphorylation prediction
to identify general probability of phosphorylation in the tested protein: NetPhos
2.0 (http://www.cbs.dtu.dk/services/NetPhos/) [20], PRedPhospho (http://pred.
ngri.re.kr/) [21], and DisPhos [22]. To identify kinase specificity we applied
NetPhosK 1.0 (http://www.cbs.dtu.dk/services/NetPhosK/) [18]. We chose to
use NetPhosK because it was the most advanced kinase-specific prediction
program available at the time of the analysis. The three-dimensional model of
protein K (see above) was used to identify those potential phosphorylation sites,
which are not buried in the protein core and thus, are possible to be modified in
the native protein.
3. Results and discussion
3.1. MS analysis of K protein sites phosphorylated in vitro
K protein molecule contains 32 serines, 24 threonines and
17 tyrosines (Accession Number P61980) (Fig. 1). 3D
modeling of K protein suggested that most of these residues
are located on the surface of the molecule and thus may
represent sites that could be phosphorylated. Analysis of K
protein amino acid sequence revealed several consensus
sequences for phosphorylation by serine-threonine kinases:
Ca2+ /Calmodulin-Dependent Protein Kinase II (CaMKII),
CK1, CK2, glycogen synthase kinase-3 (GSK3), cyclin-
dependent kinase 1 (Cdk1) (formerly known as p34cdc2), 70-
kDa ribosomal S6 kinase S6 (p70S6) and protein kinase C
(PKC) (http://www.cbs.dtu.dk/services/NetPhos/). We won-
dered how many of these sites are phosphorylated in vitro by
single enzyme.
We have previously shown that K protein is phosphorylated
in vitro by the Src-like kinase, Lck [7] and CK2 [23]. Like these
enzymes, CK1 can also potentially target several sites within K
protein. We chose CK1, CK2 and Lck to test if they
phosphorylate a single or, as predicted by computer-based
analysis, multiple sites. Bacterially expressed recombinant K
protein was purified by affinity chromatography using Ni-NTA
agarose, and the phosphorylation reaction was carried out
directly on the beads [7]. Phosphorylated proteins were
proteolytically digested with trypsin and the resulting peptides
were analyzed by mass spectrometry.
Phosphorylation sites in K protein were identified by
analysis of peptide mixtures obtained by protein K enzymatic
or chemical fragmentation. Analytical steps involve LC-ESI-
MS-MS/MS and database searching. Our identification of
phosphorylated peptides was based on the presence of an
expected 80 Da mass shift of the parent ion mass and the
analysis of MS/MS patterns of these peptides. Using both
measurements confirmed the identity of the peptide sequence.
The MS/MS CID fragmentation patterns were also analyzed in
search for precise localization of the phosphorylation site within
the peptide sequence. However, this search proved successful
only for tyrosine phosphorylated peptides, while it did not work
for all serine and threonine phosphorylated peptides. In all cases
of Ser/Thr phosphorylated peptides, the MS/MS fragmentation
spectra were identical for both non-phosphorylated and
phosphorylated versions of the peptide, in agreement with the
well documented lability of Ser/Thr phosphorylation in CID
experiment. Ser/Thr phosphorylated peptides in CID experi-
ment suffer from loss of the phosphate moiety preceding any
fragmentation of peptide bonds. This results in loss of an 80 Da
or 98 Da mass and makes fragmentation patterns of Ser/Thr
phosphorylated peptides indistinguishable from patterns
obtained for non-phosphorylated peptides. Thus, owing to

Table 1
Phosphorylated sites of K protein, identified by mass spectrometry, after in vitro phosphorylation with CK1, CK2 and Lck or phosphorylated in vivo
a.a. a.a. sequence No of S In vitro In In vitro
range and T vivo
CK 1 CK2 CK1/2 No. Lck
(No. of (No. of (No. of of Y (No. of
P-sites) P-sites) P-sites) P-sites)
1. 1–21 METEQPEETFPNTETNGEFGK 4 4 4 4 2 0 –
2. 36–46 SRNTDEMVELR 2 1 1 1 1 0 –
3. 47–52 ILLQSK 1 0 0 0 0 0 –
4. 70–86 TDYNASVSVPDSSGPER 5 2 2 2 1 1 1
5. 87–103 ILSISADIETIGEILKK 3 3 3 3 1 0 –
6. 104–139 IIPTLEEGLQLPSPTATSQLPLESDAVECLNYQHYK 6 0 0 0 1 2 2
7. 140–148 GSDFDCELR 1 0 0 0 1 0 –
8. 149–163 LLIHQSLAGGIIGVK 1 1 1 1 1 0 –
9. 167–179 IKELRENTQTTIK 3 0 0 1 0 0 –
10. 180–191 LFQECCPHSTDR 2 0 0 0 1 0 –
11. 208–219 IILDLISESPIK 2 2 2 2 1 0 –
12. 269–277 GGRPMPPSR 1 0 0 0 0 0 –
13. 278–286 RDYDDMSPR 1 1 0 1 1 1 1
14. 306–325 NLPLPPPPPPRGGDLMAYDR 0 – – – 0 1 1
15. 378–396 GSYGDLGGPIITTQVTIPK 4 2 2 2 1 1 1
16. 397–405 DLAGSIIGK 1 1 1 1 1 0 –
17. 415–422 HESASIK 2 0 0 0 0 0 –
18. 423–433 IDEPLEGSEDR 1 1 0 1 0 0 –
19. 434–456 IITITGTQDQIQNAQYLLQMSVK 4 1 1 1 0 0 –
Total 50 19 17 20 13 6 6
302 M. Mikula et al. / Biochimica et Biophysica Acta 1764 (2006) 299–306

their lability, mass spectrometry does not seem to be useful for
localization of phosphoserines and phosphothreonines.
When His-K protein was phosphorylated in vitro by Lck, 6
phosphotyrosines were identified (Table 1). For Tyr phosphor-
ylation fragmentation patterns of modified vs. unmodified
peptides differ and allow for precise localization of phosphor-
ylation sites (Fig. 2B). This is in agreement with the well
documented higher stability of Tyr phosphorylation.
The results of analysis of in vitro phosphorylation of K
protein by CK1 and CK2 are summarized in Table 1. Mass
spectral analyses revealed that in vitro CK1, CK2 and both
kinases together phosphorylate respectively 19, 17 and 20 total
serines and threonines.
3.2. MS analysis of K protein sites phosphorylated in vivo
To map sites that are phosphorylated in vivo, K protein was
purified from HTC-IR cell lysates by immunoprecipitation, and
then digested either with trypsin alone or with trypsin, V8 protease
and cyanogen bromide. The resulting peptides in the digests were
analyzed using LC-ESI-MS-MS/MS. With the three enzymes 92%
of sequence coverage of K protein was obtained.
Mass spectrometry analysis revealed 4 phosphorylated
peptides containing a unique potential phosphorylation site
each: S141, S154, S284, S401 (Table 1). In addition, six other
singly phosphorylated peptides were found, containing multiple
potential phosphorylation sites in their sequences and one
doubly phosphorylated peptide with four threonines in the
sequence. In these cases, the phosphorylation sites could not be
precisely identified. Altogether, MS analyses of endogenous K
protein's tryptic digests identified 13 phosphorylated residues,
and the same analyses performed on K protein digested with
trypsin, V8 protease and cyanogen bromide revealed 14
phosphorylated sites. Addition of a phosphate group increases
the mass of peptides by 80 Da. Since particular fragments of K
protein were represented by phosphopeptides with both one and
multiple 80 Da added to predicted mass, our MS analysis
revealed that K protein not only is phosphorylated on multiple
sites spanning its entire length but also can exist in multiple
phosphorylation states.

Fig. 2. Phosphoserine- (A) and phosphotyrosine-containing peptides (B) identified by mass spectrometry. Bacterially expressed recombinant K protein was purified by
affinity chromatography using Ni-NTA agarose, and K proteins-agarose beads were phosphorylated using CK1 (A) or Lck (B) in vitro. K protein was digested with
trypsin, and the resulting phosphopeptides were analyzed by mass spectrometry. Peptide mixture was applied to nano-HPLC RP-18 column, directly coupled to nano-
Z-spray ion source of Q-Tof electrospray mass spectrometer working in the regime of data dependent MS to MS/MS switch. The output list of parent and daughter ions
was submitted to a database search using MASCOT (MatrixScience) program. Peptide identification and the presence of their covalent modifications were verified by
inspection of parent mass fragmentation patterns using the programs MassLynx [11] and ProteinProspector. (A) An example of phosphoserine-containing peptide that
was identified based on the shifted mass value and the MS/MS sequencing pattern corresponding to identified peptide sequence. (B) An example of phosphotyrosine-
containing peptide that was identified based both on the analysis of peptide mass value and by amino acid MS/MS sequencing.
 M. Mikula et al. / Biochimica et Biophysica Acta 1764 (2006) 299–306 303

In experiments with phosphorylation in vitro we used
purified K protein. In standard in vitro phosphorylation
reactions there is one protein and one enzyme, thus, it is not
surprising that purified kinases will phosphorylate all exposed
cognate target residues. Within the cell K protein exists in
complex with RNA and other proteins. Thus, it is not
unexpected that many potential target sites are masked and
are unable to be phosphorylated with high enough stoichiom-
etry to be detected. There are sites within K protein that are
phosphorylated in vivo but are not targets of either CK1 or CK2
phosphorylated in vitro. The discrepancy between the in vitro
and in vivo results likely reflects the existence of other serine/
threonine kinases that phosphorylated K protein, which may
account for those other phospho-serine/phospho-threonine sites.
The level of K protein phosphorylation is modulated by
mitogens, cytokines and oxidative stress [4,7,8,10]. We wished
to compare how the state of K protein phosphorylation differs
between resting and proliferating cells and cells under oxidative
stress. To induce quiescence, cells were maintained in serum-
free media. Then, cells were treated with serum to induce
transition from quiescent to proliferating state while H2O2/
Na3VO4 treatment was used to induce oxidative stress. Both
mitogenic stimulation and oxidative stress increased the level of
K protein phosphorylation on tyrosine residues [7,24].
K protein immunoprecipitated from cells was separated by 2-
D gel electrophoresis and visualized by both silver stain and
immunostaining with anti-phosphotyrosine antibody. As shown
in Fig. 3, the antiphosphotyrosine blots revealed immunostain-
ing of several distinct K protein isoforms from proliferating
cells and cells under oxidative stress. While the intensity of
phosphotyrosine immunostaining was higher for all isoforms of
K protein isolated from proliferating cells as compared to
resting cells, the highest phosphorylation signals of K protein's
isoforms were obtained from cells under oxidative stress.
The 2-D gel experiments suggested that although less
intense, the pattern of K protein phosphorylation in proliferating
cells was indistinguishable from that seen in cells under
oxidative stress, suggesting that the state of K protein
phosphorylation under the two different conditions could be
similar. To test the similarities further, we digested K protein
purified from these cells with trypsin and/or V8 protease and the
resulting peptides were analyzed by LC-ESI-MS-MS/MS mass
spectrometry to map phosphopeptides. Of the same three
phosphopeptides which were isolated from both proliferating
cells and cells under oxidative stress but not from quiescent
cells, two contained tyrosines. In agreement with the 2-D gel
analysis, the percentages of peptides containing phosphotyr-
osines were higher in cells treated with H2O2/Na3VO4 than in
proliferating cells (Fig. 3). Along with the 2-D gel pattern in this
and a prior study [12], these results are consistent with a model
where K protein within the cell exists in a limited number of
phosphorylation states. With respect to these phosphorylation
compendium, there could be one set of K protein forms found in
the resting state and another one in either actively dividing cells
or cells under oxidative stress.
3.3. Structural modeling of the experimental data
We used the derived structure of K protein to map the
position of predicted as well as experimentally determined
phosphorylation sites (Fig. 4). In the case of uncertainties as to
the location of the modified residue within a peptide, we
identified the most likely sites based on the predictions made by
the NetPhos and PredPhospho servers and the location of the
side-chains in the structure. In particular, the model suggested
that phosphorylation of a number of Ser, Thr, or Tyr residues
(Y72, T96, T107 in KH1, T174, T176 in KH2, and S417, T436,
S454 in KH3 domains) is in conflict with the structure of the

Fig. 3. K protein isolated from resting, proliferating and oxidative stress-stimulated cells. The HTC-IR cells were seeded at a density of 104/cm2 in DMEM and were
grown to about 60% confluence. Cells were made quiescent by 48 h serum deprivation and then cells were either untreated or treated with 15% FBS for 24 h or with
3.5 mM H2O2 and 0.1 mM Na3VO4 for 15 min. Cells were collected, lysed in IP buffer containing protease and phosphatase inhibitors, and K protein was isolated from
sepharose beads conjugated with #54 antibody using 0.1 M glycine, pH = 2.6. Finally, K protein was separated by 2-D gel electrophoresis and visualized by both silver
stain (A) and immunostaining with anti-phosphotyrosine antibody (B). K protein from the same experiment was also digested with trypsin, V8 protease and cyanogen
bromide (C). The resulting phosphopeptides were analyzed by mass spectrometry, as described in Fig. 2.
304 M. Mikula et al. / Biochimica et Biophysica Acta 1764 (2006) 299–306

Fig. 4. A detailed map of all empirically and computationally defined K protein phosphorylation sites. Line 1: the amino acid sequence of K protein, amino acids
colored according to their physico-chemical character. Line 2: predicted secondary structure (“e”, extended/strand; “H”, helix; “x”, disordered/flexible, “−”, static
loop). Line 3: position of the side-chain in the tertiary structure (“B”, buried in the core, otherwise—partially of fully exposed). Line 4: boundaries of domains—NLS,
nuclear localization signal (blue); KH, K homology domains (green); K interactive region including RGG boxes (orange); KNS, nuclear shuttling domain (black).
Lines 5–7: location of residues predicted to be phosphorylated by NetPhos [20], NetPhosK for CKI [18], NetPhosK for CKII [18], PredPhospho-motif [21],
PredPhospho-SVM [21], DisPhos using a general model [22], and DisPhos using an Eukaryote-specific model [22] are indicated by S, T, or Y, depending on the type of
the residue. Line 13: location of phosphorylation sites identified by previous experimental analyses. Line 14: a map of peptides identified in this work by MS analysis
(indicated by “b==N”), including all theoretically possible sites of modification (indicated by “P”). Lines 15 and 16: the results of in vitro and in vivo analyses are
reconciled, with confident positions of phosphorylated residues indicated by S, T, or Y. “x” indicates the residues, for which there is no convincing experimental
evidence as for modification and which are unlikely to be modified based on the results of computational analysis (according to prediction servers and the location in
the structure). “+” indicates residues in the regions, for which no peptide data was obtained in the course of our work (hence the experimental evidence is not yet
available), but they were either found to be modified in previous works or predicted as potentially phosphorylated by at least one of the computational methods. “1”
indicates sets of residues within individual peptides, of which only one is phosphorylated, but it is unclear which one, even if the structural and computational analysis
is taken into account; “2” analogously indicates a set of sites, of which only two are phosphorylated.

unmodified protein, because the hydroxyl groups of these
residues are buried and could become accessible only upon a
conformational change. Most of these residues were regarded as
least likely to be phosphorylated and labeled as x in Fig. 4.
In one case, however, a buried residue (e.g., Y72) is found to
be phosphorylated (Table 1) [9]. The conformation of the
backbone of this residue as well as its spatial position is
modeled with high confidence. Yet, there is no rotamer that
would make the OH group of this Tyr residue fully exposed to
allow modification. We have previously shown that in K protein
phosphorylation of one site(s) enhances phosphorylation of
other sites [7]. It is conceivable that phosphorylation of Y72 is
made possible because there is significant conformational
change that is brought about by phosphorylation of other sites.
The three KH domains of K protein bind RNA synergisti-
cally [25] so that inhibition of binding by any single KH domain
can significantly diminish the binding of the protein to RNA as
a whole. Tyrosine phosphorylation inhibits binding of K protein
to RNA [7,9]. Y72 is phosphorylated in vivo and in vitro (Table
1) [9]. Preliminary modeling suggests that phosphorylation at
this position will disturb the structure predicted for the
unmodified form, probably leading to a conformational change,
 M. Mikula et al. / Biochimica et Biophysica Acta 1764 (2006) 299–306
because the phosphate group cannot be accommodated in the
protein core as it would be both sterically and electrostatically
incompatible with the surrounding residues. We suggest that the
modification of Y72 may lead to a rotation and dislocation of
helix 63–72, leading to a significant conformational change of
the GKGG loop in KH1 [26–28]. This in turn could have a
significant impact on nucleic acid binding capabilities of this
domain. Although the exact nature of the putative conforma-
tional changes cannot be inferred from the current model, this
analysis is consistent with the observations that tyrosine
phosphorylation greatly reduces the ability of K protein to
bind target RNAs [7,9].
There were also several phosphorylation sites found in the
unstructured KI region (Table 1). It has become increasing clear
that the intrinsically disordered regions are found in proteins
like K that are involved in signal transduction and gene
expression [29]. How the phosphorylation of this unstructured
segment regulates folding upon binding to its many known
partners [1] is a structural issue that remains to be addressed.
While NetPhos, DisPhos, and PredPhospho do not discrim-
inate between different kinases, NetPhosK 1.0 is a kinase-
specific predictor. It predicted 3 and 14 sites as potentially
phosphorylated by CKI and CKII, respectively. Among them,
S127 and S141 were found not to be modified, and T232, S340,
T344, S347, T351 are in regions not analyzed by MS. In
experiments performed in vitro, 19 and 17 sites were
phosphorylated by CKI or CKII (Table 1). Thus, if only the
regions analyzed by MS are considered, NetPhosK 1.0 yielded
10 true positives and 2 false positives and missed as many as 19
sites (false negatives).
The currently available prediction methods are trained on the
database of phosphorylation sites (true positives). Adding
additional sites to the database will improve the sensitivity of
these methods. More importantly, the training of computational
methods requires also true negatives (sites that are not
phosphorylated), which are very rarely reported. Adding such
sites to the database will decrease false positives predictions.
Thus, our data not only add to the knowledge about the
posttranslational modifications of the K protein, but ought to be
an important addition to the training set of computational
methods for prediction of phosphorylation sites and improve
their performance.
Although K protein has many potential phosphate acceptor
sites (Fig. 1), a single enzyme can phosphorylate several serine,
threonine and tyrosine residues (Table 1). 2-D gel electropho-
resis of K protein isolated from cells revealed 5–8 spots (Fig. 3)
[12]. Compared to quiescent cells the pattern of K protein
phosphorylation in proliferating cells and cells under oxidative
stress appears indistinguishable (Fig. 3). These results are more
consistent with a model where K protein may exist in only a few
phosphorylation states, rather than the high number predicted
by the power formula. However, the MS analysis does not
distinguish whether or not a given set of phosphopeptide digests
came from the same molecule or different molecules of K
protein. Thus, whether K protein exists in a limited or a very
large number of phosphorylation states remain unanswered.
New strategies will have to be developed to answer this critical
305
question not only to understand the structural biology of K
protein but also that of many other phosphoproteins.
K protein exits in a complex with more than one hundred
different proteins [30]. Yet, structural basis for the diversity of
its interaction is not known. Since K protein has many possible
phosphorylation sites, addition of highly charged phosphate
groups could allow K protein to be highly interactive. However,
our data favor a model in which K protein exists in a limited
number of phosphorylation states. If so, K protein phosphor-
ylation cannot by itself explain the great diversity of its
interaction, and that other mechanism likely plays a major role.
Acknowledgements
This work was supported by a grant from the Polish
Committee for Scientific Research (PBZ-KBN-039/PO4/2001)
(JO). J.O. work was also supported by a SCHOLAR GRANT
from the Foundation for Polish Science.
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