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Techniques for detecting phosphorylation sites in proteins?

Techniques for detecting phosphorylation sites in proteins?



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I would like to know how phosphorylated sites in proteins are detected in practice. I read some papers where the authors were talking about mass spectrometry techniques.

But my question is that why can't X-ray crystallography or NMR techniques capture phosphorylated sites?

(Note : My background is electronics engineering, so I am not an expert in this area.)


In principle X-ray crystallography or NMR could detect phosphorylation sites but they are much more complex and expensive techniques than mass spec. So for simply figuring out phosphorylation patterns in a protein is much easier using mass spec.

Detailed reasons:

For X-ray you need to crystallize the protein which is often very difficult/impossible and requires quite a lot of sample material. Afterwards you need a good X-ray source (ideally a synchrotron beamline) and record the scattering pattern over an extended range of angles (at least 180 degrees) before your crystal evaporates in the intense X-ray beam. Last but not least, you need additional chemical tricks, elaborate mathematical models and a large amount of computing power to compensate for the fact that in X-Ray crystallography you lose the phase information (because there are no suitable lenses for X-rays).

For NMR, you need large, very strong superconducting magnets, sophisticated radio transmitters and receivers, complicated math and computing power. However even if you have all that, you are still limited to very small proteins because otherwise the NMR spectrum is simply too complicated to be solved.

In contrast, for mass spec, you need a (highly sophisticated) spray gun, a moderate magnetic field and an electron multiplying detector. This is still a very sophisticated machine but much simpler compared to the above. In addition, data analysis is simpler than in the above cases.

Basically if you don't need to know the structural changes induced by phosphorylation (for which you would need to compare the structures of two crystals), you would stick to mass spec.


X-ray crystallography has been used to detect phosphorylated sites.

The RCSB protein database currently contains 856 structures that have both a resolution below 3 angstroms and the keyword "phosphorylated" in their listing.

It also appears to be possible to use NMR to study phosphorylated proteins.

The situations where NMR/x-ray crystallography or mass spectrometry are used differ. These differences may explain why a paper discussing the detection of phosphorylation using mass spectrometry would make no mention of NMR or x-ray crystallography.

NMR/x-ray crystallography provide structural data and almost always utilise known protein sequences.

Mass spectrometry has two major strengths for phosphorylation site detection and these highlight the reasons for using it. The first strength is the ability to gain information about many phosphorylation events, including unknown ones, from a single liquid chromatography MS/MS run. The second strength is the potential to quantify multiple phosphorylation sites from a minimum of 3 runs of a single sample.


Mapping of Phosphorylation Sites of Gel-Isolated Proteins by Nanoelectrospray Tandem Mass Spectrometry: Potentials and Limitations

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Study protein phosphorylation the old-fashioned way

One of the older methods for studying protein phosphorylation is the labeling of proteins with radiolabeled 32P-orthophosphate. The kinase will transfer the radiolabel to its substrate allowing you to detect activity. Typically, you incubate live cells with the radiolabel for a set amount of time prior to harvesting the cells, prepare cell extracts and separate the proteins by gel electrophoresis (+/- an immunoprecipitation step for a particular protein). You then dry the gel down and expose it to film or a phosphorimager screen to detect labeled bands.

Radiolabeling works well if you want to determine if your protein is phosphorylated without knowing the site of phosphorylation or the kinase involved. You can also use it in a pulse chase format or during different experimental conditions to determine when phosphorylation occurs in vivo. Because the protein is exposed to both the kinases and phosphatases, you can also get a good sense of the steady-state level of phosphorylation.

Radiolabelling tells you if your target protein is phosphorylated or gives you a general idea of kinase activity in a population, but it does not give you information about the involved kinase(s). Radiolabeling is labor intensive and of course, requires additional safety, regulatory and disposal conditions due to the radioisotopes. Also, you might need to expose your gel to film for days for adequate signal. Who has time for that?


Chemical Approaches to Studying Labile Amino Acid Phosphorylation

Phosphorylation of serine, threonine, and tyrosine residues is the archetypal posttranslational modification of proteins. While phosphorylation of these residues has become standard textbook knowledge, phosphorylation of other amino acid side chains is underappreciated and minimally characterized by comparison. This disparity is rooted in the relative instability of these chemically distinct amino acid side chain moieties, namely phosphoramidates, acyl phosphates, thiophosphates, and phosphoanhydrides. In the case of the O-phosphorylated amino acids, synthetic constructs were critical to assessing their stability and developing tools for their study. As the chemical biology community has become more aware of these alternative phosphorylation sites, methodology has been developed for the synthesis of well-characterized standards and close mimics of these phosphorylated amino acids as well. In this article, we review the synthetic chemistry that is a prerequisite to progress in this field.

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Discussion

Protein phosphorylation is one of most well-studied PTMs and is reported to be involved in regulating numerous cellular processes in prokaryotic cells 8,57 . In 2015, we collected 7,391 known p-sites of 3,750 proteins in 96 prokaryotes from published literature and developed dbPSP 1.0 22 to contain these datasets. Due to the accumulation of phosphorylation information, here we released dbPSP 2.0 by adding 11,905 new entries to include newly discovered phosphoproteins and p-sites in prokaryotes. Furthermore, the rich annotations derived from 88 public databases were integrated. In total, dbPSP 2.0 contained 19,296 known p-sites in 8,586 phosphoproteins and occupied the size of

9 GB, with a 300-fold increase compared to that of version 1.0.

In this study, to cover the diverse biological roles of prokaryotic phosphoproteins, we included multiple-layer knowledge from other databases to comprehensively annotate phosphoproteins. For example, the prokaryotic ClpP enzyme plays an important role in modulating various biological processes, such as cellular stress response, pathogenesis and homeostasis 58 . Inhibiting the function of ClpP was reported to affect the infectivity and virulence of microbial pathogens 59 . Moreover, the arginine phosphorylation of ClpP was essential for maintaining its function 20,21,60 . As shown in Fig. 6, the B. subtilis protease ClpP is annotated as a serine peptidase and participates in eliminating damaged proteins during heat shock, and its activity can be repressed by CtsR as well as by 20,697 compounds. Meanwhile, ClpP might interact with 9 partners and self-assemble in hexameric ring structures (Fig. 6). In particular, we found nearly 15,700 records from 6 orthologous databases to demonstrate that ClpP is a highly conserved subunit in prokaryotes, and the results are consistent with previous studies. In addition, the functional domain and p-site information of ClpP were also provided. In dbPSP 2.0, the curated data resources of p-sites and phosphoproteins as well as annotation information are downloadable at http://dbpsp.biocuckoo.cn/Download.php.

An overview of multiple-layer annotations for B. subtilis ClpP in dbPSP 2.0.

In summary, the dbPSP 2.0 database will be continuously maintained and updated when new p-sites in prokaryotes are identified. In addition to adding additional annotations from other public databases, we will further develop computational tools for the prediction of prokaryotic p-sites. We anticipate that this database can provide helpful support for better understanding the regulatory mechanisms and functions of phosphorylation in prokaryotes.


Protein phosphorylation is a common type of posttranslational modification (PTM) that is present in all biological species, and it affects key properties of proteins involved in numerous cellular events. In living cells, the continuous and dynamic phosphorylation and dephosphorylation of proteins at specific amino acid residues are controlled by complex signaling networks, resulting in the production of a variety of phosphoproteins with various states of phosphorylation. Protein phosphorylation occurs on several amino acid residues, including His, Asp, Glu, Lys, Arg, and Cys, on which it is very labile and difficult to detect, while more stable phosphorylation takes place on three specific residues, Ser, Thr, and Tyr. In bacterial cells, His- and Asp-phosphorylated proteins are well known for their leading roles in the two-component signal transduction system. In higher eukaryotic cells, on the other hand, Ser-, Thr-, and Tyr-phosphorylated proteins are predominant. Since the standard free energies for bonds of imidazole-phosphate on the His residue and carboxyl-phosphate on the Asp residue are large, the phosphorylated His and Asp have the potential to serve as intermediates in phosphotransfer reactions to other amino acids. Therefore, in addition to Ser-, Thr-, and Tyr-phosphorylated proteins, His- and Asp-phosphorylated proteins play crucial roles as a sensor apparatus and a response regulator, respectively, of the two-component system in quick response to intra- and extracellular signals in prokaryotes as well as in fungi and plants.

This reversible PTM is generally catalyzed by the opposing activities of large families of protein kinase and phosphatase enzymes. For example, the human genome encodes more than 500 protein kinases and about 300 protein phosphatases. Approximately 13000 human proteins have sites that are phosphorylated and dephosphorylated. These numbers reflect the importance and the complexity of protein phosphorylation. Abnormal phosphorylation resulting from an imbalance in the enzymatic reactions of kinases and phosphatases has been implicated in a wide range of human diseases, including cancer, diabetes mellitus, neurodegeneration, and immune/inflammatory and vascular disorders. Therefore, methods for quantitative and qualitative monitoring of alterations in the phosphorylation states of certain proteins are also very important for studies on the proteome, particularly in relation to the elucidation of the molecular origins of diseases and the rational molecular design of drugs.

This Special Issue will focus on the role of protein phosphorylation in all living cells. Original manuscripts and reviews dealing with any aspect of protein phosphorylation and related pathophysiology and methodology are very welcome.

Dr. Eiji Kinoshita
Dr. Emiko Kinoshita-Kikuta
Guest Editors

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Introduction

Signal transduction is the process by which a stimulus or stress is converted to a signal within cells. The signal is then conveyed to final effectors, the activity of which is necessary to generate appropriate cellular responses. Signal transmission is achieved by enabling the initial stimulus or stress to control several intermediate regulators which can simultaneously alter the functions of many targets. Among intermediate regulators, protein kinases and phosphatases have been given much attention.

Protein kinases are a large gene superfamily involved in modifying protein functions through post-translational serine/threonine/tyrosine phosphorylation. These kinases play essential roles in controlling various events such as cell division and signalling, and differentiation and metabolism ( Hanks & Hunter 1995 Nishizuka 1988, 1992 Norbury & Nurse 1992 ). In many cases, individual protein kinases are regulating multiple cellular processes through the phosphorylation of a variety of substrates, although their precise molecular mechanisms are largely unknown. Protein phosphorylation is a reversible event which is regulated by an elaborately harmonized balance between the activities of protein kinases and phosphatases ( Cohen 1989 ). As protein phosphorylation is a vital function, efforts have long been made to clarify the physiological meaning of various protein kinases and their substrates. In vitro biochemincal analyses, using purified kinases and substrates, are useful in characterizing catalytic properties of kinases and to detect phosphorylation-related functional changes of substrates. However, such analyses do not always reflect phenomena which occur in vivo.

Numerous protein kinases change subcellular location by cell signalling (for review see Inagaki et al. 1994 ). The most widely studied example is protein kinase C (PKC) which was first demonstrated biochemically to translocate from the cytosol to the membrane fraction on activation by phorbol ester (for review see Nishizuka 1995 ). These observations implied that the subcellular distribution of kinases is an important factor in determining kinase–substrate interactions in vivo. Several commonly used methods assist in investigating kinase activity in vivo. Biochemical subcellular fractionation facilitates the acquisition of spatial information concerning localization of the kinase. However, this approach does not aid in a better understanding of the in vivo kinase activity state the enzyme activity separated by this method does not always reflect in vivo activity, and technical limitations make it difficult to obtain clear images of the distribution of the kinase. To monitor in vivo kinase activity temporally, labelling of cells with radioactive phosphate is one strategy however, here too, it is difficult to acquire information on the state of spatial kinase activation. Analyses of kinase localization using specific antibodies allow for a depiction of the distribution of a ‘whole’ enzyme, which of course does not necessarily mean the ‘active’ enzyme.

Taken together, classic techniques such as biochemical cell fractionation, isotope-labelling and specific antibodies for kinases have a common serious defect the data obtained by these methods do not provide important ‘in vivo’ spatiotemporal information on ‘active’ kinases (phosphorylation/dephosphorylation state of target proteins). Up to (1990), accumulating evidence strongly suggested that the subcellular distribution of protein kinase activities is dynamically organized during various processes such as cell signalling and the cell cycle itself. Since the spatiotemporal dynamics of kinase activities appears to account for the elaborate coordination of cellular functions, a novel method addressing the above-mentioned problems was eagerly awaited. During the course of our research on intermediate filaments (IFs) we had similar setbacks, and attempts were made to establish a new approach for monitoring spatiotemporal changes of phosphorylation/dephosphorylation by in vivo kinase/phosphatase. Consequently, in 1990 we devised a method and procedures to develop site- and phosphorylation state-specific antibodies ( Yano et al. 1990, 1991 Nishizawa et al. 1990, 1991 ). As the antibodies are convenient to use, studies on cell and developmental biology, medical and biochemical fields have expanded, and numerous site- and phosphorylation state-specific antibodies against various proteins have become commercially available (for review see Inagaki et al. 1997 ).

We describe here the history and recent advances in site- and phosphorylation state-specific antibodies, as related to various cell biological and clinical events.

History of the site- and phosphorylation state-specific antibodies

Since IFs were long thought to be relatively stable compared to other cytoskeletons such as microtubules and actin filaments, it was so unexpected to find that site-specific phosphorylation induces a dramatic disassembly of vimentin filaments in vitro ( Inagaki et al. 1987 ). Since then, a similar in vitro phosphorylation-dependent disassembly was noted in many other IF proteins, including glial fibrillary acidic protein (GFAP), desmin, keratin, α-internexin, neurofilament (NF)-L and lamin (for reviews see Inagaki et al. 1996, 1997 Omary et al. 1998 ), strongly suggesting that the functions of IFs are closely involved in their phosphorylation. We carried out extensive studies to determine the physiological meaning of IF protein phosphorylation and to identify the kinases responsible for phosphorylation in vitro (for review see Inagaki et al. 1989 ). The next step was to visualize the spatiotemporal phosphorylation/dephosphorylation of IF proteins, and for this we developed site- and phosphorylation state-specific antibodies. The most important advantage of these antibodies is that a phosphorylation site can be predesigned as an epitope, by immunizing animals with a synthetic peptide containing a phosphorylated amino acid residue. (We first used as an antigen the synthetic peptide phosphorylated by purified protein kinases. There is now an established method for synthesizing phosphopeptides, without kinase.) With this method, IF studies have progressed, since this method systematically links biochemical and cell biological analyses (Fig. 1). This strategy is applicable to any protein analysis involving phosphorylation. Experimental procedures on site- and phosphorylation state-specific antibodies have been described in detail ( Inada et al. 2000 ).

Strategy for studying intermediate filament dynamics by observing phosphorylation-related events.

In next section, we will refer to examples where we analysed the site-specific phosphorylation of IF proteins and identified the responsible in vivo kinases, using site- and phosphorylation state-specific antibodies. It is noteworthy that this analysis yielded information on phosphorylation/dephosphorylation at the level of a single cell.

The potential of site- and phosphorylation state-specific antibodies for monitoring in vivo IF protein kinase activities

Site- and phosphorylation state-specific antibodies are indeed useful for in vitro studies, and have proved their worth in in vivo analyses. We have identified a number of in vitro phosphorylation sites of IF proteins, including vimentin, GFAP and desmin by various kinases such as cAMP-dependent protein kinase (PKA), PKC, Ca 2+ /calmodulin-dependent protein kinase II (CaM-K II), cdc2 kinase and Rho-kinase, and a series of antibodies recognizing phosphorylation state of respective residues were developed as listed in Table 1 (for added information see Inagaki et al. 1996, 1997 ). We found that several residues are phosphorylated by a single kinase e.g. Ser33 and Ser82 residues of vimentin are sites specific for PKC and CaM-K II, respectively. These specific sites serve as a pertinent substrate for detecting targeted kinase activity in vivo, using Western blotting or immunocytochemistry.

Protein Site Kinase Description Year Source*
GFAP Ser34 Rho kinase cleavage furrow in cytokinesis 1990,1991 1,2
Ser8 Cdc2 kinase early mitotic phase 1990,1991 3,4,5
Ser7 Rho kinase cleavage furrow in cytokinesis 1992 5
Ser13 Rho kinase cleavage furrow in cytokinesis 1992 5
synapsin I Ser9 PKA forskolin stimulation 1991 6
CaM-K II
DARPP-32 Thr34 PKA agonist-induced increase in cAMP or cGMP level 1992 7
CREB Ser133 PKA nuclear entry of PKA 1993 8
phospholamban Ser16 PKA β-adrenergic stimulation 1994 9
Thr17 CaM-K II β-adrenergic stimulation
calponin Thr184 PKC in vitro phosphorylation of free calponin 1994 10
tyrosine hydroxylase Ser40 PKA agonist-induced increase in cAMP level 1995 11
RNA polymerase II YSPTSPS motifs in carboxy-terminal report domain ? transcription on the developmental and heat shock puffs 1993 1994 12 13
erbB-2 Tyr1248 autophosphoryl. c-erbB-2 activation 1992 14
EGF stimulation 1992 15
tau Ser396 MAPK paired helical filaments in Alzheimer's disease, 1992 16,17,18,19
Ser404 MAPK foetal brain development, biopsy-derived adult brain 1994
β1-integrin toplasmic v-Src podosomes of RSV-transformed cells 1994 20
insulin receptor Ser1327 PKC major site upon phorbol ester stimulation
Thr1348 PKC phorbol ester or insulin stimulation 1994 21
vimentin Ser55 Cdc2 kinase early mitotic phase 1994 22
Ser82 CaM-K II Ca 2+ -signal dependent phosphorylation 1995 23
Ser33 PKC membrane reorganizaton during mitosis 1996 24
Ser50 PKC membrane reorganizaton during mitosis
keratin 18 Ser52 ? S amd G2/M phase 1995 25
glutamate receptor Ser696 ? AMPA stimulaton in postsynaptic densities 1995 26
CaM-K II Thr286 autophosphoryl. distributed mainly in neuronal somas other than neuropils 1993 27
GAP-43 Ser41 PKC 1994 28
GFAP Ser13 Rho-kinase occurs at cleavage furrow in cytokinesis 1996 29
Ser34 Rho-kinase occurs at cleavage furrow in cytokinesis 1996
tau Ser202 ? 1995 30
Thr205 ? 1995
N-myc S51 ERK required for the transcriptional repression activity of N-myc 1996 31
Rb Ser780 CyclinD1/CDK4 occurs in G1 phase, and the RB does not bind to E2F-1 1996 32
myelin basic protein Thr98 ERK observed in major dense line of brain white matter in vivo 1996 33
tau Ser422 ERK accumulated in neurofibrillary lesions of Alzheimer's disease 1996 34
Rb Ser608 CyclinA/CDK2 cell cycle-regulated and occurs prior to entry into S phase 1997 35
CyclinD1/CDK4
tau Ser199 ERK2 many alternate phosphorylations of sites may produce 1997 36
Ser202 ERK2 the same conformation of tau.
Thr205 ERK2
Ser235 ERK2
NMDA-R NR1 Ser890 PKC phosphorylation by PKC and PKA affects subcellular 1997 37
Ser896 PKC localization of the NR1 subunit.
Ser897 PKA
BtK Y223 autophosphoryl. serial phosphorylation of Y551 and Y223 may regulate 1997 38
Y551 Src Bruton's tyrosine kinase (BtK)
p53 Ser15 DNA-PK occurs in response to DNA damage 1997 39
Ser37 DNA-PK
p53 Ser33 CAK the phosphorylation may be involved in DNA repair 1997 40
histone H3 Ser10 ? occurs in a mitosis-specific manner 1997 41
ERK Thr202 ? Mixed lineage kinase 2 (MLK2) indirectly activates ERK 1998 42
Tyr204 ?
JNK/SAPK Thr183 MKK7 MLK2 kinase indirectly activates JNK/SAPK along 1998 42
Tyr185 MKK7 microtubules
c-jun Ser63 ? regulated by unidentified mechanisms in cerebellar neurones 1998 43
c-Src Tyr416 autophosphoryl. Src and FAK modulate Ca 2+ signalling in smooth muscle cells 1998 44
vimentin Ser71 Rho-kinase observed at cleavage furrow during cytokinesis 1998 45
S6 kinase Thr252 ? S6 kinase activity in vivo is most closely related to the Thr412 1998 46
Ser394 ? phosphorylation state
Thr412 ?
adducin PKC observed in dendritic spines 1998 47
PAK2 Thr402 autophosphoryl. the kinase activity is regulated by the phosphorylation 1998 48
aplysia elF4E Ser207 PKC 1998 49
tau Ser262 CaM-K II involved in regulation of tau/microtubule binding 1998 50
moesin Thr558 Rho-kinase important for microvilli-like structures 1998 51
p53 Ser389 ? occurs specifically with UV irradiation 1998 52
myosin light chian Ser19 MLCK induces activation of myosin ATPase 1998 53
XCAP-D2 Thr1314 Cdc2 kinase the phosphorylation may trigger mitotic chromosome 1998 54
Thr1348 condensation
Thr1353
BtK Tyr551 Src BtK signalling occurs in the region of the Ig receptor 1999 55
Tyr223 autophosphoryl. signalling complex
p53 Ser37 ATR kinase occur in DNA-damaged cells 1999 56
Ser15 ATM/ATR kinase important for p53 stabilization after DNA damage 1999 57
Ser20 ? occurs within minutes of DNA damage 1999 58
Ser15 DNA-PK not essential for activation of p53 and G1 arrest upon ionizing radiation 1999 59
Ser389 p38MAPK UV-responsive event 1999 60
Rb Ser601 ? phosphorylated in response to retinoic acid 1999 61
Ser605 ? phosphorylated in control and retinoic acid-treated cells
Ser773 ? phosphorylated in response to retinoic acid
EGF receptor Tyr1173 ? may be involved in age-associated decrease in hepatocyte proliferation 1999 62
Cdk inhibitor p27 Thr187 Cdks act as a signal for the ubiquitination of p27 1999 63
Stat3 Ser727 ? occurs by both ERK-dependent and -independent pathways 1999 64
NF-L Ser473 Casein kinase II abundant in neuronal perikarya of brain cortex 1999 65
tau Ser214 PKA observed in Alzheimer's disease brain tisues 1999 66
Ser409 PKA
caldesmon Ser759 ERK major site for ERK in carotid arteries and may have roles in 1999 67
Ser789 ERK cell division
PKCα Thr250 autophosphoryl. 1999 68
GFAP Thr7 ? occurs at cleavage furrow in cytokinesis 1999 69
histone H3 Ser28 occurs in chromosomes during early mitosis 1999 69
desmin Thr16 Rho-kinase occurs at cleavage furrow in cytokinesis 70
Thr75 Rho-kinase
Thr76 Rho-kinase
glutamate receptor 1 Ser845 PKA D1-dopamine receptor-stimulation promotes the phosphorylation 1999 71
myosin phosphatase target subunit 1 Thr695 Rho-kinase an inhibitory phosphorylation site on the molecule 1999 72
myosin binding subunit of myosin phosphatase Ser854 Rho-kinase occurs in respose to TPA- or HGF-stimulation 1999 73
FKHRL1 Thr32 Akt/PKB IGF-1 induces the phosphorylation by Akt 1999 74
Ser253
Raf Ser338 ? the phosphorylation is inhibited by Akt signalling 1999 75
Raf Ser259 Akt/PKB IGF-1 induces the phosphorylation and results in Raf inactivation 1999 76
adducin Thr445 Rho-kinase the phosphorylation inhibits the binding of adducin to F-actin 1999 77
nuclear protein ANO39 Ser145 Cdc2 kinase 2000 78
NF-L Ser55 PKA modulated by okadaic acid-sensitive phosphatase 2000 79
aquaporin-2 Ser256 PKA(?) occurs upon stimulation by arginine vasopressin 2000 80
Cdc25C Ser216 Chk2/Cds1 caffeine abolishes G2/M checkpoint by inhibition of 2000 81
Chk2/Cds1 Ser68 ATM kinase ATM kinase
SOX9 transcription factor Ser211 PKA enhance transcriptional and DNA-binding activity of SOX9 2000 82
FAK Tyr397 autophosphoryl. inhibited by Src activation 2000 83
MAP2 Thr1620 GSK-3 the phosphorylation may modify microtubule stability 2000 84
Thr1623
CPI-17 Thr38 Rho-kinase the phosphorylation increased CPI-17 activity 2000 85
CPI-17 Thr38 PKN the phosphorylation increased CPI-17 activity 2000 86
p53 Ser392 ? occurs in response UV radiation and induces the DNA-binding function of p53 2000 87
Ser20 Chk1/Cds1 Chk1 and Cds1 may play roles in regulating p53 after DNA damage 2000 88
Ser15 ATM kinase ionizing radiation enhances ATM kinase activity which phosphorylates p53 at Ser15 2000 89
Thr55 ? first report of Thr phosphorylation of p53 2000 90
Ser315 ? occurs in response to irradiation damage 2000 91
Ser46 ? occurs upon severe DNA damage by which apoptosis is induced 2000 92
myc Thr58 ? critical for determining the stability of Myc 2000 93
Ser62 ? critical for determining the stability of Myc
PAK Thr423 autophosphoryl. activated by the autophosphorylation 2000 94
PAK Thr423 PDK1 independent of PI3-kinase activity 2000 95
Waf1/Cip p21 Ser146 ? the phosphorylation modulates p21–PCNA interaction in vivo 2000 96
tau Thr212 ? doubly phosphorylated tau is accumulated in Alzheimer's 2000 97
Ser214 ? and other neurodegenerative diseases
GSK-3β Ser9 Akt/PKB induces inhibition of GSK-3β activity 2000 98
CRMP-2 Thr555 Rho-kinase induces collapse of growth cones 2000 99
MARCKS Ser159 Rho-kinase lisophosphatidic acid stimulation increased the phosphorylation 2001 100
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We developed a monoclonal antibody which specifically recognizes CaM-K II-induced vimentin phosphorylation. In combination with an antibody recognizing PKC-induced site-specific vimentin phosphorylation, CaM-K II activity was shown to be spatially regulated, since the simultaneous stimulation of endogenous PKC and CaM-K II by receptor-mediated phosphoinositide hydrolysis led to an exclusive vimentin-phosphorylation by CaM-K II, but not by PKC ( Ogawara et al. 1995 ). Thus, PKC and CaM-K II, both of which are activated through common receptors, are regulated separately, under physiological conditions, perhaps because of the different subcellular targeting of these kinases. In relation to this, it is notable that receptor-mediated Ca 2+ signalling in a localized area of an astrocyte induced CaM-K II-dependent vimentin phosphorylation in the same restricted area but not in other regions of the cell, whereas receptor stimulation sufficient to evoke Ca 2+ waves induced CaM-K II-dependent vimentin phosphorylation in the whole cell body (Fig. 2) ( Inagaki et al. 1997 ). A typical example of the application of site- and phosphorylation state-specific antibodies for spatial kinase activation is CaM-K II.

Local and global signalling of CaM kinase II defined by the area of Ca 2+ signals. (A) Local Ca 2+ signalling evokes localized signalling of CaM kinase II. a-c, [Ca 2+ ]i in an astrocyte before (a), and at 30 s (b) and 4 min (c) after the local application of 10 µ m PGF2α for 15 s. The arrow in (a) indicates the site of PGF2α application. Arrowheads in (b) indicate the process that showed Ca 2+ signalling. (d) Vimentin phosphorylation at Ser82 by CaM kinase II in the same astrocyte in (a–c). The photograph is magnified to present the area indicated by a rectangle in (b). The cell was fixed at 5 min after the [Ca 2+ ]i measurement in (c) and immunostained by MO82. Arrowheads indicate the process that involved CaM kinase II signalling. Bar = 20 µm (B) Ca 2+ wave evokes global signalling of CaM kinase II. a–c, [Ca 2+ ]i in an astrocyte before (a), and at 30 s (b), and 90 s (c) after the local application of 10 µ m PGF2α for 15 s. The arrow in (a) indicates the site of PGF2α application. d, Vimentin phosphorylation at Ser82 by CaM kinase II in the same astrocyte in (a–c). The cell was fixed at 5 min after the [Ca 2+ ]i measurement in (c). Bar = 20 µm. Modified with permission from Inagaki N. et al. (1997) .

Another example of the successful monitoring of spatiotemporal kinase activation state is Rho-kinase. We developed antibodies recognizing phosphorylated Ser7, Ser13 and Ser38 residues of GFAP ( Nishizawa et al. 1991 Matsuoka et al. 1992 Sekimata et al. 1996 ), and detected an in vivo kinase activity that phosphorylates GFAP at the cleavage furrow during cytokinesis (Fig. 3A). The kinase activity termed ‘cleavage furrow kinase (CF kinase)’ was for so long unidentified but thought to be an ubiquitous kinase. Many observations have pointed to a role of a small GTPase, Rho, in cytokinesis by inducing and maintaining the contractile ring (for review see Kaibuchi et al. 1999 Bishop & Hall 2000 ), and Rho-kinase, one Rho target, was found to phosphorylate GFAP at the same sites (Thr7, Ser13 and Ser38) as CF kinase in vitro ( Kosako et al. 1997 ). The in vitro Rho-kinase phosphorylation sites on other IF proteins, vimentin (Ser38 and Ser71) and desmin (Thr16, Thr75 and Thr76), were also shown to be phosphorylated in vivo at the cleavage furrow where Rho and Rho-kinase accumulate during cytokinesis ( Goto et al. 1998 Inada et al. 1999 ). We propose that Rho-kinase is activated in a spatiotemporal manner and facilitates efficient type III-IF segregation as a CF kinase during cytokinesis.

Site-specific phosphorylation of GFAP during mitosis and bridge-like structures of GFAP with mutations in CF kinase phosphorylation sites. (A) Metaphase or anaphase U251 cells stained with the antibody MO389 (anti-GFAP), YC10 (anti-phosphoSer8 on GFAP), TMG7 (anti-phosphoThr7 on GFAP), KT13 (anti-phosphoSer13 on GFAP), or KT34 (anti-phosphoSer38 on GFAP green). DNAs are stained with propidium iodide (red). Modified with permission from Matsuzawa et al. (1998) . (B) GFAP bridge-like structures in T24 cells expressing a mutant GFAP (Thr7, Ser13 and Ser38 are changed into Ala). Green and red colours represent GFAP stained with MO389 and propidium iodide, respectively. Bar = 10 µ m . Modified with the permission from Yasui et al. (1998) .

We then constructed a series of mutant GFAPs, where Rho kinase (CF kinase) phosphorylation sites were variously mutated, and expressed them in type III IF-negative cells. The mutations thus induced impaired the segregation of glial filaments into postmitotic daughter cells. As a result, an unusually long bridge-like cytoplasmic structure formed between the unseparated daughter cells. An alteration of other sites, including the cdc2 kinase-phosphorylation site, led to no remarkable defect in glial filament separation (Fig. 3B)( Yasui et al. 1998 ). We also demonstrated that site-specific mutations of vimentin at CF kinase (Rho kinase and Ser72-kinase) and PKC induced the formation of an IF bridge between the unseparated daughter cells ( Yasui et al. 2001 ). These findings are direct evidence that the phosphorylation of IF proteins is essential for filament segregation during mitosis.

Application of site- and phosphorylation state-specific antibodies for signal transduction and transcription studies

Some kinases, such as PKC, cdc2 kinase, mitogen-activated protein kinases (MAP kinases) and p21-activated kinase (PAK), are activated by phosphorylation at specific amino acid residues. Since site-specific phosphorylation of these kinases reflects their catalytically activated states, site- and phosphorylation state-specific antibodies against these sites may be useful for detecting active kinases. Spatiotemporal activation of PKCα in living cells was successfully imaged, using a site-specific antibody to phosphorylated Thr250, in combination with fluorescence resonance energy transfer (FRET) ( Ng et al. 1999 ). The temporal and spatial distribution of activated PAK1 following platelet-derived growth factor (PDGF) stimulation or during wound closure, was also monitored using a specific antibody against phosphorylated Thr423 ( Sells et al. 2000 ). During closure of a fibroblast monolayer wound, PAK1 was rapidly activated and was localized at the leading edge of motile cells, then gradually tapered off with wound closure.

The protein-tyrosine kinase group, first recognized among retroviral oncoproteins, includes a large number of enzymes that specifically phosphorylate Tyr residues, and are widely recognized for their roles in transducing growth and differentiation signals. Site- and phosphorylation state-specific antibodies have been developed and used for functional analyses of tyrosine kinases. In differentiated smooth muscle cells, an anti-phospho Src antibody revealed that the autophosphorylated active c-Src (P416Y) level dramatically increased following PDGF receptor stimulation. The phosphorylation is thought to be important for the association of focal adhesion kinase (FAK) and c-Src, and may modulate basal Ca 2+ channel activity and the l -type Ca 2+ current ( Hu et al. 1998 ). FAK, a nonreceptor protein tyrosine-kinase, plays a key role at cellular adhesion sites. With an antibody against autophosphorylation at Tyr397 of FAK, the possible importance of integrin and Src in FAK signalling was proposed ( McLean et al. 2000 ). As for the epidermis growth factor (EGF) receptor, an antibody against phospho-Tyr1173, which interacts with an adaptor protein Shc, was produced ( Palmer et al. 1999 ). Analyses made using this antibody demonstrated that age-dependent decreases in the ability of the EGF receptor to associate with Shc are due to differences in the Tyr-phosphorylation state of the receptor.

On the other hand, MAP kinases such as extracellular signal-regulated kinase (ERK), c-Jun-N terminal kinase (JNK)/stress-activated protein kinase (SAPK) and p38 are activated by dual phosphorylation at Thr and Tyr residues. Using synthetic diphosphopeptides as antigens, specific antibodies recognizing active dual phosphorylated MAP kinases were developed. These antibodies led to evidence that ERK translocates to the nucleus upon stimulation and is activated there ( Yung et al. 1997 ), while activated JNK/SAPK was observed along with microtubules as well as in the nucleus ( Nagata et al. 1998 ). Various kinds of anti-dualphospho-MAP kinase antibodies are commercially available and are in wide use in various researches.

A research field where site- and phosphorylation state-specific antibodies have exhibited notable power, is p53 signalling. In response to various stresses to the cell, for example DNA damage, transcription factor p53 is activated and induces cell cycle arrest and apoptosis ( Giaccia & Kastan 1998 ). Although p53 contains multiple phosphorylation sites in its N- and C-termini, the physiological significance of the phosphorylation remained largely unknown. Various Ser and Thr residues, including Ser15, Ser20, Ser33, Ser37, Ser46, Thr55, Ser315, Ser376, Ser378, Ser389 and Ser392, were found to be phosphorylated (Table 1). Phosphorylation of Ser15 and Ser20 by ataxia-telangiectasia mutated (ATM) kinase or the related kinase ATR (ATM and Rad3-related) kinase inhibits degradation of p53 through dissociation from MDM2 ( Shieh et al. 1997 Unger et al. 1999 Chehab et al. 2000 ). Ser33 was found to be phosphorylated by CDK-activating kinase (CAK) in the presence of p36 in vitro (Ko et al. 1997), while Ser37 was reported to be phosphorylated in vivo when DNA damage occurred ( Shieh et al. 1997 ). The physiological significance of these phosphorylations remains to be clarified. Ser315 of p53 is phosphorylated by CyclinB/Cdc2 or CyclinA/CDK2 in vitro, and this phosphorylation enhanced binding to the p53-responsive element ( Wang & Prives 1995 ). Ser376 and Ser378 are phosphorylated under normal conditions, and Ser376 is selectively dephosphorylated in case of DNA damage ( Waterman et al. 1998 ), 14-3-3 can bind to this region and DNA-binding ability of p53 is increased through conformational changes. It was also reported that Ser46 is phosphorylated an event noted upon DNA damage severe enough to induce apoptosis ( Oda et al. 2000 ). All these data clearly demonstrate that site- and phosphorylation state-specific antibodies contribute greatly to cancer research.

The antibody is also used in studies on medical research into Alzheimer's disease and diabetes mellitus. tau is a microtubule-binding protein which is mainly distributed in neuronal axons, and its microtubule-assembling activity is regulated by phosphorylation. To date, 9 Ser/Thr residues have been reported to be phosphorylated, determined using respective site- and phosphorylation state-specific antibodies (Table 1). It is notable that phosphorylation at Ser214 and Ser409 was seen to be increased in brain tissues from Alzheimer's patients ( Jicha et al. 1999 ). Moreover, dual phosphorylation at Thr212 and Ser214 occurs in brain tissues from Alzheimer's patients and also in other neurodegenerative diseases ( Ksiezak-Reding et al. 2000 ). Site- and phosphorylation state-specific antibodies were also used for studies on noninsulin-dependent diabetes mellitus (NIDDM). Antibodies against phospho-Ser1327 and -Thr1348 of insulin receptor were developed, and Ser1327 was seen to be phosphorylated by phorbol ester, while Ser1348 was phosphorylated upon phorbol ester and insulin stimulation ( Coghlan et al. 1994 ). These antibodies were also used for medical research on NIDDM ( Kellerer et al. 1995 ). Will phospho-specific antibodies become pertinent tools for elucidating the molecular basis of Alzheimer's disease, NIDDM and other diseases?

In addition to the examples described here, numerous antibodies recognizing the phosphorylation of a specific site(s) (see Table 1) are widely used in various researches.


Materials and Methods

Sample preparation

HEK cells were transfected with FLAG-GIT1 (4 μg per 100 mm dish) using lipofectamine. After 48 hours, cells were incubated with 1 mM peroxovanadate for 30 minutes and extracted with 25 mM Tris, 100 mM NaCl, 0.5% NP-40, pH 7.4. In some experiments, cells were incubated with 1 mM peroxovanadate and 5 nM calyculin A for 30 minutes, and then extracted. The lysates were precleared twice with mouse IgG-agarose for 1 hour at 4°C and immunoprecipiatated with FLAG-agarose (Sigma) for 2 hours at 4°C. Samples were washed twice with 25 mM Tris, 100 mM NaCl, pH 7.4 and FLAG-tagged GIT1 was eluted by incubating the beads with 0.2 mg/ml FLAG peptide in 25 mM Tris for 30 minutes at 4°C.

Sample analysis

Eluted samples were reduced and alkylated with dithiothreitol and iodoacetamide, respectively, as described by Schroeder et al. (Schroeder et al., 2004). Immunopurified amounts of GIT1 were estimated by visualization of the corresponding silver-stain band separated by SDS-PAGE. An aliquot of the immunopurified sample corresponding to an easily visible silver-stained band was digested with the desired enzyme (approximate ratio enzyme to substrate was 1:20) in 100 mM ammonium bicarbonate, pH 8.5 for 8-12 hours at room temperature. Generally, peptides from an aliquot that corresponded to 1-5% of the original immunoprecipitated sample were separated by reverse-phase chromatography using a 1-hour or 2-hour gradient as described by Schroeder et al. (Schroeder et al., 2004). Analysis of FLAG-eluted samples was with an LCQ XP or LTQ-FT (ThermoElectron, San Jose, CA) under conventional MS/MS mode according to Schroeder et al. (Schroeder et al., 2004). Reduction and alkylation steps were omitted for on-beads digestion (treatment with 100-500 ng of trypsin for 6 hours at room temperature (Schroeder et al., 2004) and analysis of the resulting peptides on an LTQ-FTMS). Enrichment of phosphopeptides was performed by immobilized metal affinity chromatography (IMAC) according to Ficarro et al. (Ficarro et al., 2002) by using 10-20× more sample. Phosphopeptides were eluted with 250 mM ascorbic acid.


Abstract

Reversible modification of proteins by phosphorylation of serine, threonine and tyrosine residues is the most common post-translational modification, which is estimated to occur in 30-90% of the cellular expressed protein component at any one time. Phosphorylation can alter proteins' subcellular distribution, enzymatic activity and specificity. Altered protein phosphorylation may correlate with disease states such as cellular transformation. The analysis of phosphorylated proteins is therefore of vital importance to the field of biology and in particular signal transduction. Protein phosphorylation sites are increasingly investigated using mass spectrometric methods, exploiting the inherent accuracy and sensitivity of these methods. However, the presence of unphosphorylated peptides in enzymatic digests of proteins causes ion suppression of phosphopeptides, reducing the effective sensitivity of detection this sensitivity is further decreased by the relative lability of the phosphate moiety in the mass spectrometer and the occurrence of sub-stoichiometric modification, which together further reduce the achievable sensitivity. This study has examined techniques for the analysis of protein phosphorylation sites, with particular emphasis upon mass spectrometry. The technique of immobilised metal ion affinity chromatography (IMAC) was investigated in detail as a method suited to phosphorylation site analysis. IMAC exploits the relatively specific affinity of phosphorylated peptides for metal ions, particularly Fe(III), to isolate phosphopeptides upon a solid-phase affinity matrix, separating the suppressing non- phosphorylated component and allowing improved detection of phosphorylated peptides. Conditions for the application of IMAC to phosphopeptide segregation have been established and applied. Using IMAC, protein phosphorylation site identification of both standards and signal transduction mediators has been carried out. Apparent sequence-specific binding of phosphorylated and non-phosphorylated peptides to IMAC resins has been found and investigated. IMAC methodology has been further improved to optimise phosphopeptide analysis using mass spectrometry. The developed methods have clear utility for phosphorylation site analysis, which is vital to the understanding of signal transduction.


Affiliations

Bioinformatics, Department of Biology, Faculty of Science, Utrecht University, Padualaan, 3584 CH, The Netherlands

Jos Boekhorst & Berend Snel

Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Sorbonnelaan, 3584, CA Utrecht, The Netherlands

Bas van Breukelen & Albert JR Heck

Academic Biomedical Centre, Utrecht University, Yalelaan, 3584, CL Utrecht, The Netherlands


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