Modulation of the G protein regulator phosducin by Ca2+/calmodulin-dependent protein kinase II phosphorylation and 14-3-3 protein binding.

Phototransduction is a canonical G protein-mediated cascade of retinal photoreceptor cells that transforms photons into neural responses. Phosducin (Pd) is a Gbetagamma-binding protein that is highly expressed in photoreceptors. Pd is phosphorylated in dark-adapted retina and is dephosphorylated in response to light. Dephosphorylated Pd binds Gbetagamma with high affinity and inhibits the interaction of Gbetagamma with Galpha or other effectors, whereas phosphorylated Pd does not. These results have led to the hypothesis that Pd down-regulates the light response. Consequently, it is important to understand the mechanisms of regulation of Pd phosphorylation. We have previously shown that phosphorylation of Pd by cAMP-dependent protein kinase moderately inhibits its association with Gbetagamma. In this study, we report that Pd was rapidly phosphorylated by Ca(2+)/calmodulin-dependent kinase II, resulting in 100-fold greater inhibition of Gbetagamma binding than cAMP-dependent protein kinase phosphorylation. Furthermore, Pd phosphorylation by Ca(2+)/calmodulin-dependent kinase II at Ser-54 and Ser-73 led to binding of the phosphoserine-binding protein 14-3-3. Importantly, in vivo decreases in Ca(2+) concentration blocked the interaction of Pd with 14-3-3, indicating that Ca(2+) controls the phosphorylation state of Ser-54 and Ser-73 in vivo. These results are consistent with a role for Pd in Ca(2+)-dependent light adaptation processes in photoreceptor cells and also suggest other possible physiological functions.

Yeast Mps1p phosphorylates the spindle pole component Spc110p in the N-terminal domain.

The yeast spindle pole body (SPB) component Spc110p (Nuf1p) undergoes specific serine/threonine phosphorylation as the mitotic spindle apparatus forms, and this phosphorylation persists until cells enter anaphase. We demonstrate that the dual-specificity kinase Mps1p is essential for the mitosis-specific phosphorylation of Spc110p in vivo and that Mps1p phosphorylates Spc110p in vitro. Phosphopeptides generated by proteolytic cleavage were identified and sequenced by mass spectrometry. Ser(60), Thr(64), and Thr(68) are the major sites in Spc110p phosphorylated by Mps1p in vitro, and alanine substitution at these sites abolishes the mitosis-specific isoform in vivo. This is the first time that phosphorylation sites of an SPB component have been determined, and these are the first sites of Mps1p phosphorylation identified. Alanine substitution for any one of these phosphorylated residues, in conjunction with an alanine substitution at residue Ser(36), is lethal in combination with alleles of SPC97, which encodes a component of the Tub4p complex. Consistent with a specific dysfunction for the alanine substitution mutations, simultaneous mutation of all four serine/threonine residues to aspartate does not confer any defect. Sites of Mps1p phosphorylation and Ser(36) are located within the N-terminal globular domain of Spc110p, which resides at the inner plaque of the SPB and binds the Tub4p complex.

Changes in protein conformational mobility upon activation of extracellular regulated protein kinase-2 as detected by hydrogen exchange.

Changes in protein mobility accompany changes in conformation during the trans-activation of enzymes; however, few studies exist that validate or characterize this behavior. In this study, amide hydrogen/deuterium exchange/mass spectrometry was used to probe the conformational flexibility of extracellular signal-regulated protein kinase-2 before and after activation by phosphorylation. The exchange data indicated that extracellular regulated protein kinase-2 activation caused altered backbone flexibility in addition to the conformational changes previously established by x-ray crystallography. The changes in flexibility occurred in regions involved in substrate binding and turnover, suggesting their importance in enzyme regulation.

Phosphorylation and subcellular redistribution of high mobility group proteins 14 and 17, analyzed by mass spectrometry.

High mobility group (HMG) proteins 14 and 17 are nonhistone nuclear proteins that have been implicated in control of transcription and chromatin structure. To examine the posttranslational modifications of HMG-14 and -17 in vivo, HMG proteins were prepared from nuclear vs. cytosolic fractions of human K562 cells treated with 12-O-tetradecanoylphorbol 13-acetate (TPA) or okadaic acid (OA) and examined by electrospray mass spectrometry. Analysis of full-length masses demonstrated mono-, di-, and triphosphorylation of HMG-14 and mono- and diphosphorylation of HMG-17 from OA treated cells, whereas HMG-14 and -17 from TPA treated cells were monophosphorylated. Peptide mass and sequence analysis showed major and minor phosphorylation sites, respectively, at Ser24 and Ser28 in HMG-17, and Ser20 and Ser24 in HMG-14. These sites were found in the consensus sequence RRSARLSAK, within the nucleosomal binding domain of each protein. A third phosphorylation site in HMG-14 was located at either Ser6 or Ser7. Interestingly, the proportion of HMG-14 and -17 found in cytosolic pools increased significantly after 1 h of treatment compared to control cells and showed preferential phosphorylation compared with proteins from nuclear fractions. These results suggest that phosphorylation of HMG-14 and -7 interferes with nuclear localization mechanisms in a manner favoring release from nuclei.

Identification of novel MAP kinase pathway signaling targets by functional proteomics and mass spectrometry.

Functional proteomics provides a powerful method for monitoring global molecular responses following activation of signal transduction pathways, reporting altered protein posttranslational modification and expression. Here we combine functional proteomics with selective activation and inhibition of MKK1/2, in order to identify cellular targets regulated by the MKK/ERK cascade. Twenty-five targets of this signaling pathway were identified, of which only five were previously characterized as MKK/ERK effectors. The remaining targets suggest novel roles for this signaling cascade in cellular processes of nuclear transport, nucleotide excision repair, nucleosome assembly, membrane trafficking, and cytoskeletal regulation. This study represents an application of functional proteomics toward identifying regulated targets of a discrete signal transduction pathway and demonstrates the utility of this discovery-based strategy in elucidating novel MAP kinase pathway effectors.

Modeling deuterium exchange behavior of ERK2 using pepsin mapping to probe secondary structure.

Recently, mass spectrometry has been applied to studies of hydrogen exchange of backbone amides, allowing analysis of large proteins at physiological concentrations. Low resolution spatial information is obtained by digesting proteins after exchange into D2O, using electrospray ionization liquid chromatography/mass spectrometry (ESI-LC/MS) to measure deuteration by mass increases of resulting peptides. This study develops modeling paradigms to increase resolution, using the signal transduction kinase ERK2 as a prototype for larger, less stable proteins. In-exchange data for peptides were analyzed by nonlinear least squares and a maximum entropy method, distinguishing amides into fast, intermediate, slow, and nonexchanging classes. Analysis of completely nonexchanging or in-exchanging peptides and peptides with sequence overlaps showed that nonexchanging amides were generally hydrogen bonded and sterically constrained or buried > or = 2.2 A from the protein surface, while fast exchanging hydrogens were generally exposed at the protein surface. In order to more fully understand the intermediate and slow exchanging classes, an empirical model was developed by analyzing published exchange rates in cytochrome c. The model correlated protection factors with a combined dependency on surface accessibility, hydrogen bond length, and position of residues from alpha helix ends. Together with analysis of partial proteolytic products, the derived rules for exchange allowed modeling of exchange behavior of peptides. Substantial deviation from the predicted rates in some cases suggested a role for conformational freedom in regulating fast and intermediate exchanging amides.

Structural characterization of the membrane-associated regulatory subunit of type I cAMP-dependent protein kinase by mass spectrometry: identification of Ser81 as the in vivo phosphorylation site of RIalpha.

The mechanism by which the type Ialpha regulatory subunit (RIalpha) of cAMP-dependent protein kinase is localized to cell membranes is unknown. To determine if structural modification of RIalpha is important for membrane association, both beef skeletal muscle cytosolic RI and beef heart membrane-associated RI were characterized by electrospray ionization mass spectrometry. Total sequence coverage was 98% for both the membrane-associated and cytosolic forms of RI after digestion with AspN protease or trypsin. Sequence data indicated that membrane-associated and cytosolic forms of RI were the same RIalpha gene product. A single RIalpha phosphorylation site was identified at Ser81 located near the autoinhibitory domain of both membrane-associated and cytosolic RIalpha. Because both R subunit preparations were 30-40% phosphorylated, this post-translational modification could not be responsible for the membrane compartmentation of the majority of RIalpha. Mass spectrometry also indicated that membrane-associated RIalpha had a higher extent of disulfide bond formation in the amino-terminal dimerization domain. No other structural differences between cytosolic and membrane-associated RIalpha were detected. Consistent with these data, masses of the intact proteins were identical by LCQ mass spectrometry. Lack of detectable structural differences between membrane-associated and cytosolic RIalpha strongly suggests an interaction between RIalpha and anchoring proteins or membrane lipids as more likely mechanisms for explaining RIalpha membrane association in the heart.

Applications of mass spectrometry to signal transduction.

Advances in mass spectrometry instrumentation, protocols for sample handling, and computational methods provide powerful new approaches to solving problems in analytical biochemistry. This review summarizes recent work illustrating ways in which mass spectrometry has been used to address questions relevant to signal transduction. Rather than encompass all of the instruments or methodologies that might be brought to bear on these problems, we present an overview of commonly used techniques, promising new methodologies, and some applications.

Mass spectral characterization of a protein-nucleic acid photocrosslink.

A photocrosslink between basic fibroblast growth factor (bFGF155) and a high affinity ssDNA oligonucleotide was characterized by positive ion electrospray ionization mass spectrometry (ESIMS). The DNA was a 61-mer oligonucleotide photoaptamer bearing seven bromodeoxyuridines, identified by in vitro selection. Specific photocrosslinking of the protein to the oligonucleotide was achieved by 308 nm XeCl excimer laser excitation. The cross-linked protein nucleic acid complex was proteolyzed with trypsin. The resulting peptide crosslink was purified by PAGE, eluted, and digested by snake venom phosphodiesterase/alkaline phosphatase. Comparison of the oligonucleotide vs. the degraded peptide crosslink by high performance liquid chromatography coupled to an electrospray ionization triple quadrupole mass spectrometer showed a single ion unique to the crosslinked material. Sequencing by collision induced dissociation (MS/MS) on a triple quadrupole mass spectrometer revealed that this ion was the nonapeptide TGQYKLGSK (residues 130-138) crosslinked to a dinucleotide at Tyr133. The MS/MS spectrum indicated sequential fragmentation of the oligonucleotide to uracil covalently attached to the nonapeptide followed by fragmentation of the peptide bonds. Tyr133 is located within the heparin binding pocket, suggesting that the in vitro selection targeted this negative ion binding region of bFGF155.

Deuterium exchange mass spectrometry as a probe of protein kinase activation. Analysis of wild-type and constitutively active mutants of MAP kinase kinase-1.

Wild-type and constitutively active mutants of human MAP kinase kinase-1 (MKK1) were analyzed by deuterium exchange mass spectrometry using a protocol that minimized loss of deuterium during analysis due to back exchange. The observed peptides accounted for 335 out of 393 residues. Not counting overlap peptides, three peptides showed decreased exchange in constitutively active compared to wild-type MKK1 and nine showed increased exchange. Backbone amides in which exchange rates decreased upon kinase activation were observed near the regulatory phosphorylation sites Ser218 and Ser222 and the adjacent beta9 strand. These decreases are consistent with electrostriction or reduced solvent access due to domain closure or formation of new hydrogen or salt bonds around the catalytic cleft and within the activation lip. Increased exchange upon activation was observed within six peptides derived from helix C and the five-stranded beta sheet from the N-proximal lobe of the conserved kinase domain and in one peptide located at the interface between the N- and C-proximal lobes. Two amides that underwent increased exchange were specifically localized between residues 68 and 69 in beta1 and 140 and 142 in beta5. These residues probably form contacts with each other on opposite sites of the beta sheet as well as with helix C. These increases appeared to represent localized fluctuations, rather than rigid body rearrangements, suggesting that MKK1 activation requires enhanced flexibility within the N-proximal lobe, perhaps to accommodate ATP binding, phosphotransfer, or ADP release.

Mass spectrometric analysis of 40 S ribosomal proteins from Rat-1 fibroblasts.

Although sequences of most mammalian ribosomal proteins are available, little is known about the post-translational processing of ribosomal proteins. To examine their post-translational modifications, 40 S subunit proteins purified from Rat-1 fibroblasts and their peptides were analyzed by liquid chromatography coupled with electrospray mass spectrometry. Of 41 proteins observed, 36 corresponded to the 32 rat 40 S ribosomal proteins with known sequences (S3, S5, S7, and S24 presented in two forms). The observed masses of S4, S6-S8, S13, S15a, S16, S17, S19, S27a, S29, and S30 matched those predicted. Sa, S3a, S5, S11, S15, S18, S20, S21, S24, S26-S28, and an S7 variant showed changes in mass that were consistent with N-terminal demethionylation and/or acetylation (S5 and S27 also appeared to be internally formylated and acetylated, respectively). S23 appeared to be internally hydroxylated or methylated. S2, S3, S9, S10, S12, S14, and S25 showed changes in mass inconsistent with known covalent modifications (+220, -75, +86, +56, -100, -117, and -103 Da, respectively), possibly representing novel post-translational modifications or allelic sequence variation. Five unidentified proteins (12,084, 13,706, 13,741, 13,884, and 34, 987 Da) were observed; for one, a sequence tag (PPGPPP), absent in any known ribosomal proteins, was determined, suggesting that it is a previously undescribed ribosome-associated protein. This study establishes a powerful method to rapidly analyze protein components of large biological complexes and their covalent modifications.

Characterization of profilaggrin endoproteinase 1. A regulated cytoplasmic endoproteinase of epidermis.

Profilaggrin, an insoluble precursor of the intermediate filament-associated protein filaggrin, contains multiple internal repeats (PIRs). At terminal differentiation of epidermis, proteolytic processing within a "linker" region of each PIR releases soluble filaggrin in a two-stage process. The first stage endoproteinase (PEP1, profilaggrin endoproteinase 1) cleaves mouse profilaggrin at a subset of the linkers, yielding processing intermediates consisting of several filaggrin repeats. An epidermal endoproteinase that cleaves the requisite linker subset has been purified 4,966-fold from mouse epidermal extracts. SDS-polyacrylamide gel electrophoresis demonstrated a band of molecular mass of 29.5 kDa that correlated with the activity. Labeling with [3H]diisopropylfluorophosphate identified PEP1 as a serine protease; inhibitor studies suggest that it is similar to chymotrypsin, as expected from previous in vivo studies. The purified PEP1 cleaved a peptide derived from profilaggrin (P1) at three residues within and adjacent to a multiple tyrosine sequence, consistent with the in vivo processing sites. No exopeptidase activity was detected. PEP1 is only active toward insoluble profilaggrin, resulting in partial solubilization, consistent with a role in dispersal of profilaggrin during terminal differentiation. In contrast to the specific cleavage of mouse profilaggrin, PEP1 cleaved all linker regions of rat profilaggrin. Studies with phosphorylated P1 suggest that PEP1 specificity may be partly regulated by profilaggrin phosphorylation.

Mass-spectrometric analysis of ADP-ribosylation factors from bovine brain: identification and evidence for homogeneous acylation with the C14:0 fatty acid (myristate).

The two proteins from bovine brain previously shown to be required for the guanosine 5'-[gamma-thio]triphosphate-dependent inhibition of a well-characterized intra-Golgi transport assay, termed GGBF and GGBF, have been definitively identified as members of the ADP-ribosylation factor (ARF) family by electrospray MS analysis of the intact proteins, and of their tryptic fragments. Extensive protein-sequence information obtained from this analysis identified GGBF and GGBF as bovine ARF1 and ARF3 respectively. The sequence of bovine ARF3, which had not previously been determined, appears identical to that predicted from the rat and human ARF3 cDNAs. Further analysis of the N-terminal tryptic fragments of both bovine ARFs demonstrates N-terminal acylation solely with the C14:0 fatty acid (myristate). This finding establishes that the previously reported specific-activity difference between ARF1 and ARF3 in the intra-Golgi transport assay is not due to lipid heterogeneity at the N-terminus. This finding also indicates that the heterogeneity of N-terminal fatty-acyl groups previously observed on other myristoylated proteins is not universal.

Mass spectrometric analysis of 21 phosphorylation sites in the internal repeat of rat profilaggrin, precursor of an intermediate filament associated protein.

Profilaggrin, a highly phosphorylated protein synthesized in mammalian cornified epithelia, is the precursor of filaggrin, a protein that is involved in aggregation of keratin during terminal differentiation. Possible functions for the phosphorylation include preventing premature aggregation of keratin, packing profilaggrin into a storage granule, association of other proteins with the granule, and/or regulating proteolytic processing of profilaggrin. As a first step in characterizing the phosphorylation of rat profilaggrin, tryptic peptides of filaggrin and profilaggrin were fractionated by reverse-phase HPLC and analyzed by ionspray mass spectrometry. Nine putative phosphopeptides were identified as those with masses 80 Da (or multiples of 80 Da) greater than the predicted unphosphorylated masses. The six that were phosphorylated to a high stoichiometry were analyzed further. Several multiply phosphorylated peptides underwent neutral loss of H3PO4 during collisional activation, complicating interpretation of the MS/MS spectra. In order to circumvent this problem, an alternative strategy was applied in which peptide mixtures were treated with Ba(OH)2, resulting in beta-elimination of H3PO4 and generation of dehydrated serine or threonine at the site of phosphorylation. Peptides containing dehydrated serine or threonine fragmented well, providing unequivocal identification of multiple phosphorylation sites in peptides as long as 39 amino acids. The phosphopeptides (with phosphorylated residues underlined) were GQQHSGHPQVYYYGVEETEDESDAQQGHHQQQQQQR, GGQAGSHSESEASGGQAGR, HTSRPEQSPDTAGR, GESPAGQQSPDR, EASASQSSDSEGHSGAHAGIGQGQTSTTHR, and GSSESQASDSEGHSDYSEAHTQGAHGGIQTSSQR.

Determination of v-Mos-catalyzed phosphorylation sites and autophosphorylation sites on MAP kinase kinase by ESI/MS.

MAP kinase kinase (MAPKK), a key component of the MAP kinase cascade, is activated through phosphorylation by several protein kinases, including the oncogene v-Mos and its cellular counterpart, c-Mos. The v-Mos-catalyzed phosphorylation sites on recombinant MAPKK1 were identified by electrospray ionization mass spectrometry as S218 and S222, located within a sequence that aligns with the T loop structure of cAMP-dependent protein kinase; these are the same as the Raf-1 phosphorylation site identified previously [Alessi, D. R., et al. (1994) EMBO J. 13, 1610-1619]. Phosphorylation of these sites was kinetically ordered, with S222 preferred over S218. Intramolecular autophosphorylation of these sites was kinetically ordered, with S222 preferred over S218. Intramolecular autophosphorylation of MAPKK occurred at several residues and was increased upon the stimulation of MAPKK activity by v-Mos. Major autophosphorylation sites were residues S298 and Y300. Minor autophosphorylation sites included T23, S299, S218, and either S24 or S25. Sequence similarities were noted between MAPKK autophosphorylation sites and exogenous phosphorylation sites on MAP kinase. Phosphorylation of either S218 or S222 was sufficient for partial MAPKK activation by Mos, and phosphorylation of S222 alone was sufficient for autophosphorylation at S298 and Y300. Mass spectral analysis was also performed on MAPKK1 purified from rabbit skeletal muscle. The peptide containing S218 and S222 was observed in only a singly phosphorylated form, and the peptide containing S298, S299, and Y300 was observed in multiply phosphorylated forms, suggesting that MAPKK is only partially phosphorylated within the T loop but significantly modified in the autophosphorylation loop under physiological conditions.

Mitogen-activated protein (MAP) kinase phosphorylation of MAP kinase kinase: determination of phosphorylation sites by mass spectrometry and site-directed mutagenesis.

Mitogen-activated protein kinase kinase (MKK) phosphorylates and activates mitogen-activated protein kinase (MAPK) in response to stimulation of various eukaryotic signaling pathways. Conversely, a recent report showed that MAPK phosphorylates MKK in vitro [Matsuda, S., Gotoh, Y., and Nishida, E. (1993) J. Biol. Chem. 268, 3277-3281]. To gain insight into the function of this feedback phosphorylation, we identified the major sites targeted for phosphorylation by MAPK and examined whether such a modification plays a role in regulating the basal and stimulated MKK activities. Two phosphopeptides generated by tryptic digestion of MAPK-phosphorylated MKK were identified by electrospray ionization mass spectrometry. Cyanogen bromide cleavage also yielded two phosphopeptides whose sequence overlapped with the tryptic phosphopeptides. Both sets of phosphopeptides contained candidate MAPK target sites at Thr292 and Thr386 that fit the consensus sequence ProXThr*Pro. Replacement of either Thr292 or Thr386 with alanine by site-directed mutagenesis reduced the phosphate incorporation respectively to 32 or 75% that of wild type MKK. Replacement of both threonine residues with alanine reduced phosphate incorporation to 2.5% that of wild type enzyme. Comparison of MAPK-phosphorylated vs. unphosphorylated MKK showed no significant differences in basal or Raf-1-stimulated MKK activity. We conclude that the phosphorylation of MKK at Thr292 and Thr386 does not interfere with catalysis in vitro.

Independent regulation of two cytoplasmic processing stages of the intermediate filament-associated protein filaggrin and role of Ca2+ in the second stage.

One of the final events in cornification of epidermal cells is processing of profilaggrin to the keratin-associated protein filaggrin. Processing involves several proteolytic events and occurs in two discrete proteolytic stages (Resing, K. A., Walsh, K. A., and Dale, B. A. (1984) J. Cell Biol. 99, 1372-1378; Resing, K. A., Walsh, K. A., Haugen-Scofield, J., and Dale, B. A. (1989) J. Biol. Chem. 264, 1837-1846). In a keratinocyte cell line derived from newborn rat epidermis, these two stages are independently regulated. Profilaggrin was expressed when the cells reached confluence; processing to intermediates began 24-36 h later (stage one), with filaggrin appearing at 48 h (stage two). Stage two processing required calcium in the medium with maximum processing occurring at 5-10 mM. Furthermore, stage two processing was inhibited by nifedipine, a calcium channel blocker, suggesting that calcium influx activates this event. Second-stage processing was also inhibited by the protease inhibitor leupeptin, implicating calpain. Confluent cells had higher levels of calpain I than subconfluent cells; in confluent cells, two immunoreactive bands were detected, comigrating with inactive (80 kDa) and activated (78 kDa) calpain I. In cells processing profilaggrin, most of the calpain I was in the 78-kDa form, implying extensive activation, supporting a role for calpain in processing.