Analysis of Phosphoprotein p19 by Liquid Chromatography/Mass

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 267, No. 5, Issue of February 15, pp. 3506-3513,1992 Printed in U. S. A.

Analysis of Phosphoprotein p19 by Liquid Chromatography/Mass Spectrometry IDENTIFICATION OF TWO PROLINE-DIRECTED SERINE PHOSPHORYLATION SITES AND A BLOCKED AMINO TERMINUS*

(Received for publication, September 4, 1991)

James E. Labdon$#, Edward NievesS, and Ulrich K. Schubartll From the Departments of $Biochemistry and llMedicine, Albert Einstein College of Medicine, Bronx, New York 10461

p19 is a highly conserved 19-kDa cytosolic protein that undergoes phosphorylation in mammalian cells upon activation of several distinct signal transduction pathways. Its expression is widespread but develop- mentally regulated. To determine the in vivo phosphorylation site(s) of p19, the protein was purified from bovine brain and resolved into the unphosphorylated form (p19) and a mixture of the two predominant phospho-forms (pp19). Proteolytic fragments of p19 and pp19 were examined by liquid chromatography/ mass spectrometry (LC/MS). We detected ion masses corresponding to fragments spanning the entire amino acid sequence as deduced from the cDNA except for those predicted to contain an unmodified amino terminus. Instead, the digests revealed ions corresponding to peptides lacking the initiator methionine and containing an N-acetylated alanine at the amino terminus. The analysis of pp19, but not that of p19, revealed two sets of ions representing peptides whose m/z values differed by 80 atomic mass units, the incremental mass of a phosphate residue. These putative phosphate-bearing peptides were sensitive to alkaline phosphatase treatment. Using combined trypsin and VS protease digestions, the phosphorylation sites were mapped to Ser-26 and Ser-38, in the peptides Leu-Ile-Leu-Ser*- Pro-Arg and Phe-Pro-Leu-Ser*-Pro-Pro-Lys, respectively. Interestingly, both phosphoserines are in a very similar sequence context, suggesting that a single proline-directed serine protein kinase, possibly p34cdc2, is responsible for phosphorylation of both sites in vivo.

Protein phosphorylation/dephosphorylation is a universal regulatory mechanism involved in signal transduction of eu- karyotic cells (Edelman et al., 1987; Hanks et al., 1988; Cohen, 1989). There is considerable interest, therefore, in identifying the protein kinase substrates that participate in the cellular signaling pathways of hormones and growth factors. One such substrate is a 19-kDa cytosolic protein, p19, that is abundant in mammalian brain and testis (Schubart, 1988; Koppel et al., 1990), where it is expressed in a developmental stage-specific manner (Amat et al., 1990, 1991). Serine phosphorylation of p19 has been demonstrated in a variety of cultured cells in response to a diverse group of extracellular factors (Brattin

* This work was supported by National Institutes of Health Grant R01 NS26333. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Regeneron Pharmaceuticals Inc. 777 Old Saw Mill River Rd., Tarrytown, NY 10591-6707.

and Portanova, 1981; Schubart, 1982; Sobel and Tashjian, 1983; Feuerstein and Cooper, 1983; Pasmantier et al., 1986; Beretta et al., 1989a; Cooper et al., 1989; Peyron et al., 1989; Sobel et al., 1989; Doye et al., 1990).

Based on the nucleotide sequence of available cDNA clones, it is now established that p19 (Schubart et al., 1989) is identical to stathmin (Doye et al., 1989) and also to p18, a protein found in human leukemic cells (Zhu et al., 1989). Others have referred to the same protein as prosolin (Brav- erman et al., 1986), pp2O/pp21/pp23 (Peyron et al., 1989) and 19K (Gullberg et al., 1990). The gene encoding p19 has been highly conserved in vertebrates. Thus, between the amino acid sequences of p19 predicted from the cDNAs of rat (Schu- bart et al., 1989; Doye et al., 1989) and man (Zhu et al., 1989), respectively, 148 of 149 positions are identical. In addition, the single amino acid difference is a conservative substitution (Val versus Ile, respectively), indicating that the structure of p19 is essentially identical in the two species. This remarkable degree of inter-species conservation strongly suggests that the protein serves an important function in mammalian cells.

Because of the diversity of extracellular signals that pro- mote p19 phosphorylation in intact cells, both CAMP-dependent protein kinase and protein kinase C have been implicated in this process. In addition, the recent demonstration of nerve growth factor-stimulatedphosphorylation of p19 in PC12 cells (Doye et al., 1990) suggests the involvement of yet another protein kinase or protein phosphorylation cascade. It has been shown that p19 can serve as a substrate for CAMP-dependent protein kinase in vitro (Schubart et aL, 1987; Beretta et al., 198913). More recently, the protein was also purified from human T lymphocytes and phosphorylated in vitro using partially purified protein kinase C (Gullberg et al., 1990). It is, therefore, of interest to identify the kinase(s) that phosphorylate(s) p19 in vivo. With this objective in mind, we have, in the present study, determined the sites at which the protein is phosphorylated in vivo. Using LC/MS’ to analyze proteolytic fragments of the unphosphorylated and phosphorylated forms of p19, isolated from bovine brain, we have identified two previously unsuspected proline-directed serine phosphorylation sites.

EXPERIMENTAL PROCEDURES

Materials

Acetonitrile was obtained from Burdick and Jackson, glycerol from Fisher, trifluoroacetic acid from Pierce Chemical Co., TPCK-trypsin

The abbreviations used are: LC, liquid chromatography; MS, mass spectrometry; FAB, fast atom bombardment; HPLC, high-perform- ance liquid chromatography; CAD, collision-activated dissociation; TPCK, ~-l-tosylamido-2-phenylethyl chloromethyl ketone.

3506

p19 Phosphorylation Sites 3507 (206 units/mg) from Worthington, V8 protease from Boehringer Mannheim, and bacterial alkaline phosphatase (100 units/pl) from GIBCO/Bethesda Research Laboratories.

Methods Purification of pl9-The unphosphorylated form (p19) and the

combined phospho-forms (pp19) of p19 were purified from bovine brain as described previously (Schubart et al., 1987). The purified proteins were lyophilized before being subjected to two-dimensional electrophoresis or enzymatic digestions.

Two-dimemiom1 Electrophoresis-Two-dimensional electrophoresis was performed as described previously (Schubart, 1982). The second dimension slab gels contained 15% acrylamide. Prior to load- ing to the isoelectric focusing gels, marker proteins were added to the samples (see legend to Fig. 1).

Enzymatic Digestions-To obtain tryptic fragments, 50 pg of p19 or pp19, and 1 pg of TPCK-trypsin were incubated in 50 pl 0.1 M NH,HC03, pH 7.9, a t 37 "C. Following overnight incubation, an additional 1 pg of enzyme was added and the incubation continued for another 24-30 h. Prior to analysis by LC/MS, the samples were stored at -20 "C. Digestion of sample with V8 protease alone was performed as described for trypsin. For combined tryptic/V8 digests, 20 pl of the original tryptic digest sample were combined with 1 pg of V8 protease and incubated as described above. To dephosphorylate putative phosphate-bearing tryptic fragments, 10 pl of the original tryptic digest sample of pp19 were combined with 1 p1 of bacterial alkaline phosphatase and incubated at 37 "C for 1 h. The sample was stored at -20 "C prior to analysis by LC/MS.

Liquid Chromatography/Mass Spectrometry-Mass spectral anal- yses were performed on a Finnigan-MAT 90 two-sector double- focusing instrument. Sample desorption and ionization was accom- plished with an 8-keV neutral Xenon beam from an Ion-Tech saddle- field FAB source. Mass calibrations were performed with cesium iodide and glycerol. Scans were acquired from 400 to 3000 atomic mass units a t a scan speed of 15 s/decade. Source temperature was maintained at 70 "C throughout the run. The mass spectrometer was used at a resolution of 1400 with an accelerating voltage of 5 kV. Solvent delivery was provided by an Applied Biosystems 130 dual- syringe pump HPLC. An Aquapore OD-300 (50 X 1.0 mm; 7 pm) microbore column was used for peptide separations and eluate was directed to a modified Finnigan-MAT Bioprobea continuous-flow LC/ MS interface. The standard MAT-90 stainless steel target for the flowprobe was modified to accept a 2.0 micron stainless steel frit (Valco 2FR2, 1/8 inch diameter X 1/32 inch thick) (Labdon et al., 1991). A fused silica capillary (0.25 mm outer diameter, 0.075 mm inner diameter) was connected to a second Rheodyne injector located on the flowprobe using Upchurch zero dead volume fittings. The target end of the capillary was passed through the center of a septum and abutted against the frit. The flowprobe was inserted into the source of the mass spectrometer to allow the target to come to temperature. After approximately 10 min, the HPLC column was connected to the flowprobe, and the apparatus was allowed to equil- ibrate for several minutes. An aliquot (5 or 10 pl) of an enzymatic digest of p19 was then injected onto the column. A 30-min gradient of 0-70% acetonitrile in 2.5% aqueous glycerol, 0.1% trifluoroacetic acid was used to separate the peptides at a flow rate of 10 pllmin. The low flow rate used in this analysis caused long delays in the elution of the peptides. Consequently, the scans were acquired for at least one hour after injection in order to ensure that all eluted peptides were analyzed.

Mass Spectral Data Analysis-The enzymatic fragmentation pat- terns of p19 were analyzed using FRAGIT, a computer program developed in our laboratory. The program accepts a text file of the sequence of a protein and calculates the exact masses and sequence positions of both complete and incomplete products of digestion by any enzyme or chemical reagent or combination thereof. The program was modified to predict the masses of all potential phosphorylated peptides. Only peptides containing either Ser, Thr, or Tyr residues were considered candidate peptides for the phospho-form calcula- tions. The assignment of sequences to ions with unpredicted m/z values was aided by another program created in our laboratory. This program, MASSGUESS, accepts an m/z value identified from the spectrum, along with a mass error (usually 1 atomic mass unit), and lists all possible sets of contiguous amino acids from the protein sequence that fulfill these two criteria, regardless of cleavage site. This program is especially useful for assigning masses to peptides generated by unusual cleavages, without regard to the specificity of

the enzyme used. For example, some products of V8 protease digestion involve cleavage at Asp, even though the enzyme has a preference for Glu residues under the conditions used.

RESULTS

Purification of p19 andppl9-As isolated from tissues and cells, p19 consists of a mixture of three major isoforms that can be resolved by two-dimensional electrophoresis (Schubart and Danoff, 1987; Schubart et al., 1987; Beretta et al., 1989b). Previous evidence suggests that they represent an unphosphorylated form, PI 6.2, and two predominant phospho-forms, PI 5.8 and PI 5.6, respectively. Thus, in cultured cells exposed to 32Pi only the more acidic forms incorporate 32P. Peptide maps suggested that the three forms are isoelectric variants of a single protein (Schubart and Danoff, 1987; Beretta et al., 198913). The three forms were shown to copurify on hydrophobic chromatography, gel filtration, sucrose density gradient centrifugation, and reverse-phase fast protein liquid chromatography (Schubart et al., 1987). The in vitro transla- tion product of p19 mRNA comigrated on two-dimensional electrophoresis with the least acidic form (Schubart, 1988). Finally, in uitro phosphorylation of this form gave rise to the more acidic forms (Schubart et al., 1987; Beretta et al., 1989b) and the latter were nearly completely converted to the former by treatment with alkaline phosphatase (Beretta et al., 1989b). We will henceforth refer to the least acidic form as p19 and to the more acidic variants collectively as pp19. Additional minor, more acidic isoforms, which are thought to represent more highly phosphorylated species, have been observed in stimulated cells by 32P autoradiography (Brattin and Portan- ova, 1981; Schubart, 1982; Sobel and Tashjian, 1983; Pasman- tier et al., 1986; Schubart et al., 1987; Toutant and Sobel, 1987; Beretta et al., 1989a, 1989b Chneiweiss et al., 1989; Peyron et al., 1989; Gullberg et al., 1990). The isoform com- position of our preparations was analyzed by two-dimensional electrophoresis using comigration with standard proteins (Fig. 1).

*/ ,, , "I . "7 i r . F . ' * ~ W W m . . ;

FIG. 1. Analysis of purified p19 by two-dimensional electrophoresis. The unphosphorylated form ( A ) and the combined phospho-forms of p19 ( B ) were purified from bovine brain (see "Experimental Procedures") and analyzed by two-dimensiuonal electrophoresis using comigration with standard proteins (bovine serum albumin, M, 67,000, PI 6.4, (upper arrowhead) and @-lactoglobulin, M , 18,000, PI 5.0 (lower arrowhead). I, unphosphorylated form (~19) ; 2 and 3, predominant phospho-forms (pp19).

3508 p19 Phosphorylation Sites

1 4

RASGQAFELILSPRSKESVPEFPLSPPKKKDLSLEE

FIG. 2. Sequence of p19. Upper, complete sequence as predicted from the cDNA. Residues in boxes are predicted sequences for phosphorylation by CAMP-dependent protein kinase. Underlined residues are predicted sequences for phosphorylation by protein kinase C (see "Discussion"). The actual sites of phosphorylation are indicated by asterisks. Numbering of the residues assumes an NHz-terminal Met residue, although we have obtained evidence that the sequence begins with an acetylated alanine. hwer,fragmentation strategy for the determination of the phosphorylation sites of pp19. Peptides were generated by trypsin (T4 and T6), V8 protease (V4 and V6), or a combination of the two enzymes (VT7 and VT11). The detection of molecular ions for all six peptides as their phospho-forms was the basis for the mapping experiments in this study.

LC/MS Analysis-The amino acid sequence of p19, predicted from the cDNA, is shown in Fig. 2. The protein consists of 149 residues and has a calculated molecular weight of 17,303. Trypsin (cleavage at Lys, Arg), V8 protease (cleavage a t Glu), and alkaline phosphatase digests of p19 were used to map covalent modification sites to specific peptides.

Tryptic Digest ofpl9"Table IA shows the mass determinations of the tryptic peptides of pp19. Almost all of the predicted fragments were observed at their expected m/z values, with the exception of the NHz- and COOH-terminal peptides. The COOH-terminal peptide was observed, however, as an incomplete digestion product (T32-33) with a weak ion (m/z 1307). This may be explained by the fact that T33 has five acidic side chains and only one basic site, the amino terminus, and would be expected to produce a weak ion under positive ion FABMS conditions. The incomplete digestion product contains an extra basic site (Lys-140) for protonation to the molecular ion.

Although the amino acid sequence predicted from the cDNA encoding p19 indicates an NH2-terminal Met residue, we did not observe an ion (m/z 980) expected for the NH2-terminal tryptic nonapeptide. Since sequencing experiments had suggested that the protein was blocked (Schubart et al., 1989), we examined the spectra for masses which would result from the most common types of blocking groups (N-formyl and N- acetyl, see Wold, 1981). Ions corresponding to complete or incomplete digestion products of a blocked NHz terminus were not observed. However, we did detect a strong ion (m/z 889) consistent with an acetylated peptide comprised of amino acids 2-9 (see below, Fig. 8). No other combination of contiguous amino acids in the sequence of p19, regardless of cleavage site, was found that could produce an ion of this mass. Digestion of p19 by other enzymes also produced ions consistent with a blocked amino terminus beginning with N- acetyl Ala instead of Met (see below).

The LC/MS analysis of the tryptic digest of pp19 produced two pairs of ions (m/z 1389/1469 and 1326/1406) which differed by 80 atomic mass units, suggesting mixtures of unphosphorylated and monophosphorylated products. The

ESWETPLSPPK 58:lE

+ P i

10 20 30 40 5 0 80 70 80 B O

Time (min)

FIG. 3. Reversed-phase LC/MS analysis of the tryptic digest of pp19. Selected ion chromatograms of the peptides V4 ( m / z 13271, V6 (m/z 1389), and their phospho-forms, pV4 (m/z 1407) and pV6 (m/z 1469) respectively. For HPLC conditions, see "Experimental Procedures." Elution time is indicated above each peak. Selected ion chromatogram plots in this paper include signals within k1.0 atomic mass unit of the selected mass and, unless otherwise indicated, full scale intensity is arbitrarily assigned to the most intense peak in each plot.

1407 1 I 1469

1 ESWETPLSPPK I I

31 38 +Pi

ASGQATELILSPR

25

MIZ

FIG. 4. FABMS spectra of tryptic peptides of pp19. Scans corresponding to the peaks for peptides T6, pT6, and T4 in Fig. 3 were accumulated and background-corrected. The inset shows the spectrum of pT4, a weak ion which was accumulated separately and displayed with a different intensity scale. Both spectra share the same m/z axis.

ions were located by selected ion chromatograms (Fig. 3), and the spectra comprising the chromatographic peaks are shown in Fig. 4. p19 was also digested with trypsin and analyzed, along with a similar digest of a second preparation of pp19. Portions of the selected ion chromatograms of the resulting fragments are shown in Fig. 5. The left panels show the peaks, derived from the digest of pp19, resulting from T4, T6, and their monophosphate forms, pT4 and pT6, respectively. The right panels show the corresponding selected ion chromatograms for tryptic peptides arising from p19. It is noteworthy that no peaks were observed for the monophosphate forms of T4 and T6, even though the intensities of the ions representing their unmodified forms were severalfold greater than in the MS analysis of pp19 (see different scales in left and right panels). The accumulated spectra of the peaks in the left panels clearly showed the presence of the ions corresponding to the phospho-forms of T4 and T6, whereas no such ions were observed in similar scans from the MS analysis of p19

p19 Phosphorylation Sites

TABLE I Mass determinations of proteolytic fragments of p19 and pp19

Theoretical m/z values are the exact masses of the peptides plus a proton for formation of the molecular ion, (M+H)+. Masses for fragments below 400 are not included. A, results for the tryptic digest (T peptides), B, the combined V8 protease/tryptic digest (VT peptides), C, the V8 protease digest alone (V peptides). Ions in boldface type represent phosphopeptide masses and are predicted to be 80 atomic mass units higher than the unphosphorylated peptides.

3509

Peptide mlz Sequence

Theoretical Observed Observed + Pi position Sequence

A T1 T2 T4 T6 T9 T11 T14 T15 T17 T18 T19 T20 T21 T22 T23 T24 T25 T29 T33 T1" T32-33b

B VT1 VT6 VT7 VTlO VT11 VT14 VT27 VT34 VT37 VT41 VT47 VT61 VT1"

C v1 v 3 v 4 v 5 V6 V8 v11 V13 V14 V17 v19 v2 1 v22 V23 V24 V25 V26 V28 V1"

978.5 518.3

1388.8 1326.7 1074.6 817.4 912.5 588.3 542.3 616.4

1165.6 607.3 498.3 592.3 418.2 748.4 417.3 782.4 962.4 889.4

1306.5

978.5 709.3 698.5 431.2 785.5 576.3 460.2 487.3 723.3 498.3 619.3 546.2 889.4

1107.5 993.5 1042.6 431.2

1598.9 758.5 812.5 800.5 432.3 800.5

1054.5 886.5 461.2 418.2 861.5 573.4 769.4 773.5

1018.5

518.3 1388.5 1326.3 1074.4 817.3 912.3 588.4 542.3 616.4

1166.2 607.5 498.3 592.4 418.2 748.3 417.2 782.5

889.3 1306.5

709.3 698.4

785.4 576.4

487.5

498.4 619.5

889.34

993.5 1042.5 431.1

1600.0 758.5 812.4 800.5 432.2 800.5

886.3

418.1 861.4 573.4

773.5 1018.5

1-9 10-13

1468.9 15-27 1406.3 30-41

44-52 54-60 63-70 71-75 77-80 81-85 86-95 96-100

101-104 105-109 110-112 113-119 120-122 129-134 141-149

2-9 136-149

1-9 15-21 22-27 31-34

778.5

865.4 35-41 44-48 71-74 82-85 90-95

101-104 114-119 141-145

2-9

1-10 13-21

1122.7 22-30

1679.4 31-34 35-48 50-55 60-65 68-74 75-77 82-88 90-98

100-106 107-110 111-113 114-121 122-125 126-131 133-138

MASSDIQVK ELEK ASGQAFELILSPR ESVPEFPLSPPK DLSLEEIQK LEAAEER SHEAEVLK QLAEK EHEK EVLQK AIEENNNFSK MAEEK LTHK MEANK ENR EAQMAAK LER HIEEVR DPADETEAD Acetyl-ASSDIQVK ESKDPADETEAD

MASSDIQVK ASGQAFE LILSPR SVPE FPLSPPK DLSLE QLAE VLQK NNNFSK LTHK AQMAAK DPADE Acetyl-ASSDIQVK

MASSDIQVKE KRASGQAFE LILSPRSKE SVPE FPLSPPKKKDLSLE IQKKLE RRKSHE VLKQLAE KRE VLQKAIE NNNFSKMAE KLTHKME ANKE NRE AQMAAKLE RLRE KDKHIE VRKNKE

2-10 Acetyl-ASSDIQVKE a Determined as an acetylated NHZ-terminal peptide.

Incomplete digestion product.

(data not shown). This result supports the notion (Schubart an aliquot of the original tryptic digest of pp19 was treated et al., 1987) that the charge differences between the isoforms with alkaline phosphatase and subjected to LC/MS analysis of p19 result from different degrees of phosphorylation, spot for a second time. The ions at m/z 1389 and 1326 (unphos- 1 representing the unphosphorylated form, and spots 2 and 3 phorylated T4 and T6, respectively) were again observed in the mono- and bis-phospho-forms, respectively (see Fig. 1). this sample. However, the ions corresponding to the phos-

Alkaline Phosphatase Treatment-In order to verify that phorylated peptides were no longer detected (data not shown). the ions at m/z 1469 and 1406 resulted from phosphorylation, This finding strongly suggests that the ions at m/z 1469 and


1.8- -3.8

5 0 6 0 7 0 Time (mln)

FIG. 5. Comparative selected ion chromatograms of tryptic digests of p l 9 and pp19. Left panels show T4, T6, and their phospho-forms pT4 and pT6 derived from pp19. Right panels show the same chromatographic regions for the analysis of the tryptic digest of p19. Neither pT4 nor pT6 were detected in this sample.

100- LILSRR I I 5 1 : s

ID. 25

m/a:llS

m/r:866 Pi I

WLSVRX 100-

50-

60: M

IO 20 30 40 50 60 m Time (mln)

FIG. 6. Selected ion chromatograms of VS/tryptic fragments of pp19. The tryptic digest sample from Fig. 3 was subjected to further cleavage by V8 protease and analyzed by LC/MS in order to determine which serines were modified. The selected ion chromatogram plots show VT7 (m/z 699), pVT7 (m/z 779), VTl l (m/z 786), and pVTll (m/z 865). The latter two represent phosphorylations at Ser-25 and Ser-38, respectively. The ion at m/z 697.5 was identified as a peptide resulting from cross-cleavage of the two enzymes (data not shown).

1406, observed in the tryptic digests of pp19, correspond to monophosphate derivatives of the tryptic peptides T4 and T6, respectively (Table IA).

V8 ProteaselTryptic Digest of pl9-Both tryptic peptides described above contain two potential phosphorylation sites. These are Ser-16 and Ser-25 in peptide T4, and Ser-31 and Ser-38 in peptide T6, respectively (see Fig. 2 and Table IA). To determine which of the Ser residues was phosphorylated, the original tryptic digest sample of pp19 was further digested with V8 protease. Cleavage of peptides T4 and T6 by V8 protease was predicted to result in four peptides, VT6, VT7, VT10, and VT11, each containing a single Ser residue. Fig. 6 shows the selected ion chromatograms for the LC/MS analysis of the combined V8/trypsin digest of pp19. Ions for peptides VT6, VT7 and VTl l were detected, but VTlO was not. VTlO would not be expected to produce a strong ion in positive-ion FABMS because of the presence of an acidic residue (Glu) and lack of a basic side chain. Two phosphopeptides, pVT7 and pVTll were detected as strong ions (Fig. 7). In contrast, ions corresponding to peptides pVT6 or pVTlO were not detected (Table IB). These results suggest that the

19

Y)

mlz

FIG. 7. FABMS spectra of peptides from a combined VS/ tryptic digest of pp19. Accumulated scans for VT7 and pVT7 (m/ z 699 and 779, respectively) from Fig. 6 are shown. The intenslty scale was multiplied 5-fold for m/z values above 750. The inset spectrum depicts accumulated spectra for VTll and pVTll (m/z 786 and 866, respectively). Peptides pVT7 and pVTll contain phospho- serine residues at Ser-25 and Ser-38, respectively. No phospho-forms of peptides corresponding to Ser-16 or Ser-31 were detected in this sample.

phosphorylation sites of p19 are Ser-25 and Ser-38. With respect to pVT7, there is no incomplete or unusual cleavage of p19 that can produce a peptide with an m/z of 779. In contrast, an incomplete cleavage product (peptide VT56, Arg- Ala-Ser-Gly-Gln-Ala-Phe-Glu) would be expected to have a m/z value similar to that of pVTll (865.4). However, there was no evidence of an incomplete digestion product involving Argl4, based on the analysis of the tryptic digest of this sample. Furthermore, the loss of phosphate from peptide T4 following alkaline phosphatase treatment is consistent with phosphorylation of either VT6 or VT7. Since no ion corresponding to pVT6 was observed, the assignment of pVT7 to m/z 866 agrees with the results of the alkaline phosphatase treatment. The ion assigned to the acetylated NH2 terminus, m/z 890, was unchanged following V8 protease treatment, which was expected, since there are no Glu residues within this peptide.

V8 Protease Digest of p19-pl9 and pp19 were also cleaved with V8 protease alone, which generated a set of peptides that overlapped the sequences of the tryptic peptides and allowed further confirmation of the assignments made in the first two experiments. The results obtained with pp19 are shown in Table IC. The mass spectral data confirm phosphorylations at Ser25 and Ser38, since peptides V4 and V6 were detected at their predicted m/z values plus 80. Although these peptides each contain two potential Ser phosphorylation sites, the only Ser residues these peptides have in common with T4 and T6 are those at positions 25 and 38 (Fig. 2). In accordance with the results of the tryptic digest, the LC/MS analysis of the V8 protease digest of p19 did not show the monophosphate derivatives of V4 and V6, further supporting the conclusion that p19 (spot 1) is the unphosphorylated protein (data not shown). The ion at m/z 1019 (Vl) appears to result from cleavage of the protein at GlulO, and confirms the presence of an N-acetylated peptide composed of residues 2-10 (Fig. 8). No other combination of contiguous amino acids from the sequence of p19, regardless of cleavage site, was predicted to have this mass.

p19 Phosphorylation Sites 3511

100

80

- %

v) c c

FIG. 8. FABMS spectra of N-acet- E 60 ylated amino-terminal fragments of - p19. Accumulated scans of the predicted g N-acetylated amino-terminal tryptic ( l e f t ) and V8 (inset) fragments 4c are shown. U

2c

701

0 II

H3C-C-N-ASSDIQVK-COOIi

0 H3C-C-N-ASSDIQvrcE-COOH

1019

1 i c

DISCUSSION

The use of LC/MS in this study offered several advantages over conventional FABMS peptide mapping analysis. It has been reported that the modification of peptides by the incor- poration of phosphate reduces their ion abundance in FABMS (Naylor et al., 1986). There are at least two reasons for the reduction in signal intensity for these peptides. The first is the negative charge added to the peptide by the phosphate moiety, which tends to reduce its ion abundance in positive- ion FABMS. The second is the ion suppression effect, which results from the clustering of hydrophobic peptides and phosphopeptides at the surface of the sample matrix droplet. Continuous-flow LC/MS is a dynamic procedure in which continuous mixing of the sample occurs at the target, thus reducing ion suppression. The use of the flowprobe also allows a significant reduction in the amount of glycerol in the solvent, thereby reducing interference by matrix ions (Caprioli et al., 1987, 1989). Using LC/MS we were also able to isolate only those scans which displayed weak ions and subtract the background, thereby improving the signal-to-noise character- istics of these ions. This allowed us to observe some peptides (e.g. pT4) that were not detected by conventional FABMS analysis (not shown).

Other laboratories have determined the location of phosphorylated residues by subjecting the molecular ion of the phosphopeptide to collision-activated dissociation (CAD) analysis (Erickson et al., 1990; Gibson et al., 1987). CAD spectra give sequence-specific ions indicating the position of the phosphate-bearing residue, as well as ions resulting from loss of phosphate from the parent. In our experiments, we did not observe ions resulting from neutral loss of phosphoric acid (-98 atomic mass units) or HP04 (-96 atomic mass units) in the spectra of the p19 phosphopeptides. Further- more, the low abundance of phosphopeptide ions in our study ruled out CAD-linked scan analysis for sequence-specific ions. For this reason, we confirmed the presence of the phosphate moiety by subjecting the tryptic digest of pp19 to alkaline phosphatase digestion.

One possible interpretation of the similar retention times of some peptide-phosphopeptide pairs on reversed-phase HPLC in this study (for example, see Fig. 3) is that the unphosphorylated peptide (m/ z -80) is actually an ion resulting from loss of HP03 from the phosphopeptide parent ion. However, it has been reported that ions from loss of

HP03 are of very low abundance in peptides containing phos- phoserine, even under CAD conditions (Gibson et al., 1987). We have consistently seen small, but significant, retention time differences between these pairs after repeated runs, indicating that they are discrete peptides.

Our analysis has identified a blocked NH2 terminus and two phosphorylation sites of p19. Although we did not find evidence for other modifications of the protein, a small frac- tion of p19 phosphorylated at sites other than the ones we have identified could have escaped detection by LC/MS. The two phosphorylation sites we have identified do not coincide with the sites we had predicted previously. Thus, although p19 contains three sites (see Fig. 2) that resemble the consen- sus sequence for substrates of CAMP-dependent protein kinase (Arg-Arg-X-Ser-X, see Edelman et d., 1987), we failed to detect the expected phosphopeptides and, instead, observed strong ions corresponding to the unmodified peptides containing these sequences. The data suggest the possibility that p19 may not serve as a direct substrate for this enzyme in viuo. However, it is also possible that phosphorylation at the predicted sites was not observed, because at the time of tissue removal, CAMP-dependent protein kinases were not activated in the cells expressing p19. Most studies utilizing 32P labeling and autoradiography have revealed, in addition to the two major phospho-forms analyzed in the present study, one or more minor more acidic 32P-labeled isoforms of p19. This finding suggests strongly that the protein contains at least one additional site that can be phosphorylated in intact cells. Our finding of only two sites suggests the possibility that phosphorylation of p19 in vivo is not a random event but proceeds in a cooperative manner. In this case, occupancy of the two sites described here would be a prerequisite for phosphorylation at one or more additional site(s). Studies are in progress to test this hypothesis.

Evidence has also been presented that p19 can serve as a substrate, both in vivo and in vitro, for protein kinase C. In the protein kinase C substrate sites identified to date, the phosphorylated Ser or Thr is generally followed by one or more basic amino acid residues (House et al., 1987; Graff et al., 1989; Noland et al., 1989). By these criteria, both Ser residues we have shown to be phosphorylated can be considered potential sites for phosphorylation by protein kinase C (see Fig. 2). Studies are in progress to determine whether these sites can serve as substrates for this enzyme in uitro.


TABLE I1 Comparison of phosphorylation sites of p19 with sequences of phosphorylation sites of other proteins

The phosphorylated or presumed phosphorylated residue of each phosphoprotein is indicated in the first column. The amino acid sequences are shown in single letter code. Boldface letters, identity with the query sequence or conservative substitutions; asterisk, phosphoamino acid; ?, protein kinase not yet identified; ??, site not yet shown to be phosphorylated; Refs.; 1, Stein et al., 1988; 2, Peter et al., 1990; Ward and Kirschner, 1990; 3, Haycock, 1990; 4, Bodwell et al., 1991; 5, Vulliet et al., 1989; 6, Cisek and Cordon, 1989; 7, McVey et al., 1989; 8, Erickson et al., 1990; 9, Countaway et al., 1989. MAP, mitogen-associated protein.

PhosDhoDrotein Protein kinase Sequence Ref.

A site 1 (Ser-25) p19, Ser-25 p19, Ser-38 SCG10, Ser-73 SCG10, Ser-62 Lamin, Ser-392 Tyrosine hydroxylase, Ser-31 Glucocorticoid receptor, Ser-234 Lamin, Ser-22 Tyrosine hydroxylase, Ser-8 RNA polymerase 11, multiple SV40 large T, Thr-124 Glucocorticoid receptor, Thr-159

B: site 2 (Ser-38) p19, Ser-38 Lamin, Ser-22 SV40 large T, Thr-124 p19, Ser-25 SCG10, Ser-73 Myelin basic protein, Thr-97 EGF receptor, Thr-669 SCG10, Ser-62 RNA polymerase 11, multiple

The fact that both phosphorylated Ser residues of p19 have a Pro residue as their COOH-terminal neighbor is of interest because the cell cycle-regulated protein kinase ~ 3 4 ' ~ " ~ phos- phorylates substrates exclusively on sites containing Ser/Thr- Pro (for reviews, see Nurse, 1990; Moreno and Nurse, 1990; Lewin, 1990). As shown in Table 11, some of the sites phosphorylated by ~ 3 4 " ~ " ' quite closely resemble the sequences of the two phosphorylation sites of p19. In addition, similar sites can be phosphorylated by other kinases (Table 11), which include a Pro-directed Ser/Thr protein kinase (Vulliet et aL, 1989) and a mitogen-activated protein kinase (Erickson et al., 1990).

The enzyme(s) that catalyze the phosphorylation of p19 in vivo remain(s) to be identified. The striking similarity between the two sites we have identified suggests that a single kinase may be responsible for phosphorylating both sites. It is noteworthy that sequences closely resembling these sites are also found in SCGlO (see Table 11), a neuron-specific protein (Stein et al., 1988) related to p19 (Schubart et al., 1989). Although phosphorylation of SCGlO has not yet been reported, it would be interesting to determine if SCGlO and p19 are phosphorylated by the same enzyme(s).

Another interesting aspect of the two phosphorylation sites determined in this study is their location within the protein. According to the secondary structure prediction rules of Chou and Fasman (1974), p19 would consist of two a-helical regions that are separated from each other by a segment of 15 amino acids, which contains 5 Pro and 4 Ser residues and would be predicted to consist of a random coil (Schubart et al., 1989). The 2 phosphorylated Ser residues we have identified are located at each end of this segment. Introducing negative charges at these positions might cause significant conforma- tional changes within the protein. The sites in SCGlO that closely resemble the phosphorylation sites of p19 (Table 11) are located in nearly identical positions relative to p19.

In conclusion, using LC/MS analysis, we have identified

? ? ?? ?? p34cdc2 ? ? p34cdc2 (?) Pro-directed p34cdc2 p34cdc2 ?

? p34cdc2 ? p34cdc2 ? ?? MAP kinase ? ?? p34cdc2

L I L S * P R S K F P L S * P P K K T L A S P K K K L P P S P I S E L S P S ' P T S Q A V T S * P R F I N L L S ' P L A G T P L S * P T R I S A P S * P Q P K P S Y S * P T S P Q H S T * P P K K G C A T * P T E K

F P L S * P P K K T P L S * P T R I Q H S T * P P K K L I L S * P R S K T L A S P K K K T P R T * P P P P E P L T ' P S G E L P P S P I S E S P T S * P S Y S

1 1 2 3 4 2 5 6 7 4

2 7

1 8 9 1 6

two sites within the amino acid sequence of p19 that undergo phosphorylation i n uiuo. We have not ruled out the formal possibility that phosphorylation at these positions occurs constitutively. However, because of the large body of evidence indicating that phosphorylation of p19 is regulated by a variety of extracellular factors, we favor the hypothesis that the two sites are involved in these regulatory events. Identi- fication of the kinase(s) that phosphorylate these sites both in uitro and i n uiuo should provide important insights regard- ing the signal transduction pathway(s) involved.

Acknowledgments-We wish to thank Dr. Richard M. Caprioli for critically reviewing the manuscript and Eric Wexler for devising the computer programs used in the data analysis.

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Documents

Analysis of Phosphoprotein p19 by Liquid Chromatography/Mass