Unraveling molecular complexity of phosphorylated human cardiac troponin I by top down electron capture dissociation/electron transfer dissociation mass spectrometry

Cardiac troponin I (cTnI), the inhibitory subunit of the thin filament troponin-tropomyosin regulatory complex, is required for heart muscle relaxation during the cardiac cycle. Expressed only in cardiac muscle, cTnI is widely used in the clinic as a serum biomarker of cardiac injury. In vivo function of cTnI is influenced by phosphorylation and proteolysis; therefore analysis of post-translational modifications of the intact protein should greatly facilitate the understanding of cardiac regulatory mechanisms and may improve cTnI as a disease biomarker. cTnI (24 kDa, pI approximately 9.5) contains twelve serine, eight threonine, and three tyrosine residues, which presents a challenge for unequivocal identification of phosphorylation sites and quantification of positional isomers. In this study, we used top down electron capture dissociation and electron transfer dissociation MS to unravel the molecular complexity of cTnI purified from human heart tissue. High resolution MS spectra of human cTnI revealed a high degree of heterogeneity, corresponding to phosphorylation, acetylation, oxidation, and C-terminal proteolysis. Thirty-six molecular ions of cTnI were detected in a single ESI/FTMS spectrum despite running as a single sharp band on SDS-PAGE. Electron capture dissociation of monophosphorylated cTnI localized two major basal phosphorylation sites: a well known site at Ser(22) and a novel site at Ser(76)/Thr(77), each with partial occupancy (Ser(22): 53%; Ser(76)/Thr(77): 36%). Top down MS(3) analysis of diphosphorylated cTnI revealed occupancy of Ser(23) only in diphosphorylated species consistent with sequential (or ordered) phosphorylation/dephosphorylation of the Ser(22/23) pair. Top down MS of cTnI provides unique opportunities for unraveling its molecular complexity and for quantification of phosphorylated positional isomers thus allowing establishment of the relevance of such modifications to physiological functions and disease status.     

Protein kinase CK2 differentially phosphorylates maize chromosomal high mobility group B (HMGB) proteins modulating their stability and DNA interactions.

The high mobility group (HMG) proteins of the HMGB family are architectural factors in eukaryotic chromatin, which are involved in the regulation of various DNA-dependent processes. We have examined the post-translational modifications of five HMGB proteins from maize suspension cultured cells, revealing that HMGB1 and HMGB2/3, but not HMGB4 and HMGB5, are phosphorylated by protein kinase CK2. The phosphorylation sites have been mapped to the acidic C-terminal domains by analysis of tryptic peptides derived from HMGB1 and HMGB2/3 using nanospray ion trap mass spectrometry. In native HMGB1, Ser(149) is constitutively phosphorylated, whereas Ser(133) and Ser(136) are differentially phosphorylated. The functional significance of the CK2-mediated phosphorylation of HMGB proteins was analyzed by circular dichroism measurements showing that the phosphorylation increases the thermal stability of the HMGB proteins. Electrophoretic mobility shift assays demonstrate that the phosphorylation reduces the affinity of the HMGB proteins for linear DNA. The specific recognition of DNA minicircles is not affected by the phosphorylation, but a different pattern of protein-DNA complexes is formed. Collectively, these findings show that phosphorylation of residues within the acidic C-terminal domain of the HMGB proteins can modulate protein stability and the DNA binding properties of the HMGB proteins.     

Mass spectrometric analysis of the kinetics of in vivo rhodopsin phosphorylation

On stimulation, rhodopsin, the light-sensing protein in the rod cells of the retina, is phosphorylated at several sites on its C terminus as the first step in deactivation. We have developed a mass spectrometry-based method to quantify the kinetics of phosphorylation at each site in vivo. After exposing either a freshly dissected mouse retina or the eye of an anesthetized mouse to a flash of light, phosphorylation and dephosphorylation reactions are terminated by rapidly homogenizing the retina or enucleated eye in 8 M urea. The C-terminal peptide containing all known phosphorylation sites is cleaved from rhodopsin, partially purified by ultracentrifugation, and analyzed by liquid chromatography coupled with mass spectrometry (LCMS). The mass spectrometer responds linearly to the peptide from 10 fmole to 100 pmole. The relative sensitivity to peptides with zero to five phosphates was determined using purified phosphopeptide standards. High pressure liquid chromatography (HPLC) coupled with tandem mass spectrometry (LCMS/MS) was used to distinguish the three primary sites of phosphorylation, Ser 334, Ser 338, and Ser 343. Peptides monophosphorylated on Ser 334 were separable from those monophosphorylated on Ser 338 and Ser 343 by reversed-phase HPLC. Although peptides monophosphorylated at Ser 338 and Ser 343 normally coelute, the relative amounts of each species in the single peak could be determined by monitoring the ratio of specific daughter ions characteristic of each peptide. Doubly phosphorylated rhodopsin peptides with different sites of phosphorylation also were distinguished by LCMS/MS. The sensitivity of these methods was evaluated by using them to measure rhodopsin phosphorylation stimulated either by light flashes or by continuous illumination over a range of intensities.     

Mass spectrometric characterization of oat phytochrome A: isoforms and posttranslational modifications.

At least four mRNAs for oat phytochrome A (phyA) are present in etiolated oat tissue. The complete amino acid sequences of two phyA isoforms (A3 and A4) and the N-terminal amino acid sequence of a third isoform (A5) were deduced from cDNA sequencing (Hershey et al., 1985). In the present study, heterogeneity of phyA on a protein level was studied by tryptic mapping using electrospray ionization mass-spectrometry (ESIMS). The total tryptic digest of iodoacetamide-modified phyA was fractionated by gel filtration chromatography followed by reversed-phase high-performance liquid chromatography. ESIMS was used to identify peptides. Amino acid sequences of the peptides were confirmed or determined by collision-induced dissociation mass spectrometry (CID MS), MS/MS, or by subdigestion of the tryptic peptides followed by ESIMS analysis. More than 97% of the phyA3 sequence (1,128 amino acid residues) was determined in the present study. Mass-spectrometric analysis of peptides unique to each form showed that phyA purified from etiolated oat seedling is represented by three isoforms A5, A3, and A4, with ratio 3.4:2.3:1.0. Possible light-induced changes in phytochrome in vivo phosphorylation site at Ser7 (Lapko VN et al., 1997, Biochemistry 36:10595-10599) as well at Ser17 and Ser598 (known as in vitro phosphorylation sites) were also analyzed. The extent of phosphorylation at Ser7 appears to be the same for phyA isolated from dark-grown and red-light illuminated seedlings. In addition to Ser7, Ser598 was identified as an in vivo phosphorylation site in oat phyA. Ser598 phosphorylation was found only in phyA from the red light-treated seedlings, suggesting that the protein phosphorylation plays a functional role in the phytochrome A-mediated light-signal transduction.     

Phosphorylation, but not alternative splicing or proteolytic degradation, is conserved in human and mouse cardiac troponin T

Cardiac troponin T (cTnT), the tropomyosin binding subunit of the troponin complex, plays a pivotal regulatory role in the Ca(2+)-mediated interaction between actin thin filament and myosin thick filament. The post-translational modifications (PTMs) and alternative splicing of cTnT may represent important regulatory mechanisms of cardiac contractility. However, a complete characterization of PTMs and alternatively spliced isoforms in cTnT present in vivo is lacking. Top-down protein mass spectrometry (MS) analyzes whole proteins, thus providing a global view of all types of modifications, including PTMs and sequence variants, simultaneously in one spectrum without a priori knowledge. In this study, we applied an integrated immunoaffinity chromatography and top-down MS approach to comprehensively characterize PTMs and alternatively spliced isoforms of cTnT purified from healthy human and wild-type mouse heart tissue. High-resolution Fourier transform MS revealed that human cTnT (hcTnT) and mouse cTnT (mcTnT) have similar phosphorylation patterns, whereas higher molecular heterogeneity was observed for mcTnT than hcTnT. Further MS/MS fragmentation of monophosphorylated hcTnT and mcTnT by electron capture dissociation and collisionally activated dissociation unambiguously identified Ser1 as the conserved in vivo phosphorylation site. In contrast, we identified a single spliced isoform for hcTnT but three alternatively spliced isoforms for mcTnT. Moreover, we observed distinct proteolytic degradation products for hcTnT and mcTnT. This study also demonstrates the advantage of top-down MS/MS with complementary fragmentation techniques for the identification of modification sites in the highly acidic N-terminal region of cTnT.     

Reversible phosphorylation at the C-terminal regulatory domain of p21(Waf1/Cip1) modulates proliferating cell nuclear antigen binding

The p53-inducible gene product p21(WAF1/CIP1) plays a critical role in regulating the rate of tumor incidence, and identifying mechanisms of its post-translational regulation will define key pathways that link growth control to p21-dependent tumor suppression. A eukaryotic cell model system has been developed to determine whether protein kinase signaling pathways that phosphorylate human p21 exist in vivo and whether such pathways regulate the binding of p21 to one of its key target proteins, proliferating cell nuclear antigen (PCNA). Although human p21 expressed in Sf9 cells is able to form a complex with human PCNA, the inclusion of cell-permeable phosphatase inhibitors renders p21 protein inactive for PCNA binding. The treatment of this inactive isoform of p21 with alkaline phosphatase restores its binding to PCNA, suggesting that p21 expressed in Sf9 cells is subject to reversible phosphorylation at a key regulatory site(s). A biochemical approach was subsequently used to map the phosphorylation sites within p21, whose modification in vitro can inhibit p21-PCNA complex formation, to the C-terminal domain at residues Thr(145) or Ser(146). A phospho-specific antibody was developed that only bound to full-length p21 protein after phosphorylation in vitro at Ser(146), and this reagent was further used to demonstrate that the inactive isoform of p21 recovered from Sf9 cells treated with phosphatase inhibitors had been phosphorylated in vivo at Ser(146). These data identify the first phosphorylation site within the C-terminal regulatory domain of p21 whose modification in vivo modulates p21-PCNA interactions and define a eukaryotic cell model that can be used to study post-translational signaling pathways that regulate p21.     

Identification of a novel phosphorylation site in c-jun directly targeted in vitro by protein kinase D

Protein kinase D (PKD) phosphorylates the c-jun amino-terminal in vitro at site(s) distinct from JNK [C. Hurd, R.T. Waldron, E. Rozengurt, Protein kinase D complexes with c-jun N-terminal kinase via activation loop phosphorylation and phosphorylates the c-jun N-terminus, Oncogene 21 (2002) 2154-2160], but the sites have not been identified. Here, metabolic (32)P-labeling of c-jun protein in COS-7 cells indicated that PKD phosphorylates c-jun in vivo at a site(s) between aa 43-93, a region containing important functional elements. On this basis, the PKD-mediated phosphorylation site(s) was further characterized in vitro using GST-c-jun fusion proteins. PKD did not incorporate phosphate into Ser63 and Ser73, the JNK sites in GST-c-jun(1-89). Rather, PKD and JNK could sequentially phosphorylate distinct site(s) simultaneously. By mass spectrometry of tryptic phosphopeptides, Ser58 interposed between the JNK-binding portion of the delta domain and the adjacent TAD1 was identified as a prominent site phosphorylated in vitro by PKD. These data were further supported by kinase reactions using truncations or point-mutations of GST-c-jun. Together, these data suggest that PKD-mediated phosphorylation modulates c-jun at the level of its N-terminal functional domains.     

Sites phosphorylated in myosin light chain in contracting smooth muscle

Purified smooth muscle myosin light chain can be phosphorylated at multiple sites by myosin light chain kinase and protein kinase C. We have determined the sites phosphorylated on myosin light chain in intact bovine tracheal smooth muscle. Stimulation with 10 microM carbachol resulted in 66 +/- 5% monophosphorylated and 11 +/- 2% diphosphorylated myosin light chain after 1 min, and 47 +/- 4% monophosphorylated and 5 +/- 2% diphosphorylated myosin light chain after 30 min. Myosin heavy chain contained 0.06 +/- 0.01 mol of phosphate/mol of protein which did not change with carbachol. At both 1 and 30 min the monophosphorylated myosin light chain contained only phosphoserine whereas the diphosphorylated myosin light chain contained both phosphoserine and phosphothreonine. Two-dimensional peptide mapping of tryptic digests of monophosphorylated and diphosphorylated myosin light chain obtained from carbachol-stimulated tissue was similar to the peptide maps of purified light chain monophosphorylated and diphosphorylated, respectively, by myosin light chain kinase; these maps were distinct from the map obtained with tracheal light chain phosphorylated by protein kinase C. Phosphorylation of tracheal smooth muscle myosin light chain by myosin light chain kinase yields the tryptic phosphopeptide ATSNVFAMFDQSQIQEFK with S the phosphoserine in the monophosphorylated myosin light chain and TS the phosphotreonine and phosphoserine in the diphosphorylated myosin light chain. Thus, stimulation of tracheal smooth muscle with a high concentration of carbachol results in formation of both monophosphorylated and diphosphorylated myosin light chain although the amount of diphosphorylated light chain is substantially less than monophosphorylated light chain. In the intact muscle, myosin light chain is phosphorylated at sites corresponding to myosin light chain kinase phosphorylation.     

Casein kinase II phosphorylates p34cdc2 kinase in G1 phase of the HeLa cell division cycle.

The activity of p34cdc2 kinase is regulated in the phases of vertebrate cell cycle by mechanisms of phosphorylation and dephosphorylation. In this paper, we demonstrate that casein kinase II (CKII) phosphorylates p34cdc2 in vivo and in vitro at Ser39 during the G1 phase of HeLa cell division cycle. Human p34cdc2 shows a typical phosphorylation sequence motif site for CKII at Ser39 (ES39EEE). In our experiments, either p34cdc2 expressed and purified from bacteria or p34cdc2 immunoprecipitated from HeLa cells enriched in G1 by elutriation were substrates for in vitro phosphorylation by CKII. Phosphoamino acid analysis, N-chlorosuccinimide mapping, and two-dimensional tryptic mapping of p34cdc2 phosphorylated in vitro were performed to determine the phosphorylation site. A synthetic peptide spanning residues 33-50 of human p34cdc2, including the CKII site, was used to map the site. In addition, phosphorylation at Ser39 also occurs in vivo, since p34cdc2 is phosphorylated during G1 on serine, and its two-dimensional tryptic map shows two phosphopeptides that comigrate exactly with the synthetic peptides used as standard.     

Regulation of protein kinase D by multisite phosphorylation. Identification of phosphorylation sites by mass spectrometry and characterization by site-directed mutagenesis

Activation of the serine/threonine kinase, protein kinase D (PKD/PKC mu) via a phorbol ester/PKC-dependent pathway involves phosphorylation events. The present study identifies five in vivo phosphorylation sites by mass spectrometry, and the role of four of them was investigated by site-directed mutagenesis. Four sites are autophosphorylation sites, the first of which (Ser(916)) is located in the C terminus; its phosphorylation modifies the conformation of the kinase and influences duration of kinase activation but is not required for phorbol ester-mediated activation of PKD. The second autophosphorylation site (Ser(203)) lies in that region of the regulatory domain, which in PKC mu interacts with 14-3-3tau. The last two autophosphorylation sites (Ser(744) and Ser(748)) are located in the activation loop but are only phosphorylated in the isolated PKD-catalytic domain and not in the full-length PKD; they may affect enzyme catalysis but are not involved in the activation of wild-type PKD by phorbol ester. We also present evidence for proteolytic activation of PKD. The fifth site (Ser(255)) is transphosphorylated downstream of a PKC-dependent pathway after in vivo stimulation with phorbol ester. In vivo phorbol ester stimulation of an S255E mutant no longer requires PKC-mediated events. In conclusion, our results show that PKD is a multisite phosphorylated enzyme and suggest that its phosphorylation may be an intricate process that regulates its biological functions in very distinct ways.     

Phosphorylation and mutations of Ser(16) in human phenylalanine hydroxylase. Kinetic and structural effects

Phosphorylation of phenylalanine hydroxylase (PAH) at Ser(16) by cyclic AMP-dependent protein kinase is a post-translational modification that increases its basal activity and facilitates its activation by the substrate l-Phe. So far there is no structural information on the flexible N-terminal tail (residues 1-18), including the phosphorylation site. To get further insight into the molecular basis for the effects of phosphorylation on the catalytic efficiency and enzyme stability, molecular modeling was performed using the crystal structure of the recombinant rat enzyme. The most probable conformation and orientation of the N-terminal tail thus obtained indicates that phosphorylation of Ser(16) induces a local conformational change as a result of an electrostatic interaction between the phosphate group and Arg(13) as well as a repulsion by Glu(280) in the loop at the entrance of the active site crevice structure. The modeled reorientation of the N-terminal tail residues (Met(1)-Leu(15)) on phosphorylation is in agreement with the observed conformational change and increased accessibility of the substrate to the active site, as indicated by circular dichroism spectroscopy and the enzyme kinetic data for the full-length phosphorylated and nonphosphorylated human PAH. To further validate the model we have prepared and characterized mutants substituting Ser(16) with a negatively charged residue and found that S16E largely mimics the effects of phosphorylation of human PAH. Both the phosphorylated enzyme and the mutants with acidic side chains instead of Ser(16) revealed an increased resistance toward limited tryptic proteolysis and, as indicated by circular dichroism spectroscopy, an increased content of alpha-helical structure. In agreement with the modeled structure, the formation of an Arg(13) to Ser(16) phosphate salt bridge and the conformational change of the N-terminal tail also explain the higher stability toward limited tryptic proteolysis of the phosphorylated enzyme. The results obtained with the mutant R13A and E381A further support the model proposed for the molecular mechanism for the activation of the enzyme by phosphorylation.     

Identification of a phosphorylation site in the hinge region of the human progesterone receptor and additional amino-terminal phosphorylation sites

We have previously reported the identification of seven in vivo phosphorylation sites in the amino-terminal region of the human progesterone receptor (PR). From our previous in vivo studies, it was evident that several phosphopeptides remained unidentified. In particular, we wished to determine whether human PR contains a phosphorylation site in the hinge region, as do other steroid receptors including chicken PR, human androgen receptor, and mouse estrogen receptor. Previously, problematic trypsin cleavage sites hampered our ability to detect phosphorylation sites in large incomplete tryptic peptides. Using a combination of mass spectrometry and in vitro phosphorylation, we have identified six previously unidentified phosphorylation sites in human PR. Using nanoelectrospray ionization mass spectrometry, we have identified two new in vivo phosphorylation sites, Ser(20) and Ser(676), in baculovirus-expressed human PR. Ser(676) is analogous to the hinge site identified in other steroid receptors. Additionally, precursor ion scans identified another phosphopeptide that contains Ser(130)-Pro(131), a likely candidate for phosphorylation. In vitro phosphorylation of PR with Cdk2 has revealed five additional in vitro Cdk2 phosphorylation sites: Ser(25), Ser(213), Thr(430), Ser(554), and Ser(676). At least two of these, Ser(213) and Ser(676), are authentic in vivo sites. We confirmed the presence of the Cdk2-phosphorylated peptide containing Ser(213) in PR from in vivo labeled T47D cells, indicating that this is an in vivo site. Our combined studies indicate that most, if not all, of the Ser-Pro motifs in human PR are sites for phosphorylation. Taken together, these data indicate that the phosphorylation of PR is highly complex, with at least 14 phosphorylation sites.     

Identification of in vivo phosphorylation sites of MLK3 by mass spectrometry and phosphopeptide mapping

MLK3 is a serine/threonine protein kinase that functions as an upstream activator of the JNK pathway. Previous work has suggested that MLK3 is a multiphosphorylated protein. In this study, mass spectrometry coupled with comparative phosphopeptide mapping was used to directly characterize MLK3 in vivo phosphorylation sites. Various types of mass spectrometry were used to analyze MLK3 tryptic peptides separated by C18 reverse-phase HPLC, leading to the identification of Ser(524), Ser(654), Ser(705), Ser(740), Ser(758), Ser(770), Ser(793), and a site found on peptide Ser(11)-Arg(37) within a Gly-rich region as MLK3 phosphorylation sites. Additionally, porous graphitic carbon chromatography successfully retained and resolved phosphopeptides that had eluted along with nonvolatile salts and buffers in the flowthrough fractions from the C18 column. Following resolution by PGC chromatography, MALDI-MS in conjunction with alkaline phosphatase treatment identified Ser(555), Ser(556), Ser(724), and Ser(727) as sites of phosphorylation on MLK3. A proline residue immediately follows 7 of the 11 unambiguously identified phosphorylation sites, suggesting that MLK3 may be a target of proline-directed kinases. Finally, two-dimensional phosphopeptide mapping confirmed that phosphorylation of Ser(555) and Ser(556) of MLK3 is induced by the activated small GTPase Cdc42.     

Multi-site phosphorylation of the protein tyrosine phosphatase, PTP1B: identification of cell cycle regulated and phorbol ester stimulated sites of phosphorylation.

The non-transmembrane protein tyrosine phosphatase, PTP1B, comprises 435 amino acids, of which the C-terminal 114 residues have been implicated in controlling both localization and function of this enzyme. Inspection of the sequence of the C-terminal segment reveals a number of potential sites of phosphorylation. We show that PTP1B is phosphorylated on seryl residues in vivo. Increased phosphorylation of PTP1B is seen to accompany the transition from G2 to M phase of the cell cycle. Two major tryptic phosphopeptides appear in two-dimensional maps of PTP1B from mitotic cells. One of these comigrates with the peptide generated following phosphorylation of PTP1B in vitro at Ser386 by the mitotic protein Ser/Thr kinase p34cdc2:cyclin B. The site of phosphorylation that is responsible for the pronounced retardation in the electrophoretic mobility of PTP1B from mitotic cells has been identified by site directed mutagenesis as Ser352. The identify of the kinase responsible for this modification is presently unknown. We also show that stimulation of HeLa cells with the phorbol ester TPA enhances phosphorylation of PTP1B. Two dimensional phosphopeptide mapping reveals that the bulk of the phosphate is in a single tryptic peptide. The site, identified as Ser378, is also the site of phosphorylation by protein kinase C (PKC) in vitro. Thus the TPA-stimulated phosphorylation of PTP1B in vivo appears to result directly from phosphorylation by PKC. The effect of phosphorylation on the activity of PTP1B has been examined in immunoprecipitates from TPA-treated and nocodazole-arrested cells. TPA treatment does not appear to affect activity directly, whereas the activity of PTP1B from nocodazole-arrested cells is only 70% of that from asynchronous populations.     

Phosphorylation of RhoB by CK1 impedes actin stress fiber organization and epidermal growth factor receptor stabilization

RhoB is a small GTPase implicated in cytoskeletal organization, EGF receptor trafficking and cell transformation. It is an immediate-early gene, regulated at many levels of its biosynthetic pathway. Herein we show that the serine/threonine protein kinase CK1 phosphorylates RhoB in vitro but not RhoA or RhoC. With the use of specific CK1 inhibitors, IC261 and D4476, we show that the kinase phosphorylates also RhoB in HeLa cells. Mass spectrometry analysis demonstrates that RhoB is monophosphorylated by CK1, in its C-terminal end, on serine 185. The substitution of Ser185 by Ala dramatically inhibited the phosphorylation of RhoB in cultured cells. Lastly we show that the inhibition of CK1 activates RhoB and promotes RhoB dependent actin fiber formation and EGF-R level. Our data provide the first demonstration of RhoB phosphorylation and indicate that this post-translational maturation would be a novel critical mechanism to control the RhoB functions.     

Regulation of CK2 by phosphorylation and O-GlcNAcylation revealed by semisynthesis

Protein serine-threonine kinase casein kinase II (CK2) is involved in a myriad of cellular processes including cell growth and proliferation through its phosphorylation of hundreds of substrates, yet how CK2 function is regulated is poorly understood. Here we report that the CK2 catalytic subunit CK2α is modified by O-linked β-N-acetyl-glucosamine (O-GlcNAc) on Ser347, proximal to a cyclin-dependent kinase phosphorylation site (Thr344). We use protein semisynthesis to show that phosphorylation of Thr344 increases the cellular stability of CK2α by strengthening its interaction with Pin1, whereas glycosylation of Ser347 seems to be antagonistic to Thr344 phosphorylation and permissive to proteasomal degradation. By performing kinase assays with site-specifically phospho- and glyco-modified CK2α in combination with CK2β and Pin1 binding partners on human protein microarrays, we show that the kinase substrate selectivity of CK2 is modulated by these specific post-translational modifications. This study suggests how a promiscuous protein kinase can be regulated at multiple levels to achieve particular biological outputs.