Identification of substrate specificity determinants for the cell cycle ...

THE JOURNAL OF B I O ~ I C C AH LE M I ~ Y 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 9, Issue of March 4, pp. 6603-6607, 1994 Printed in U.S.A.

Identification of Substrate Specificity Determinants for the Cell Cycle-regulated NIMA Protein Kinase* (Received for publication, September 10, 1993, and in revised form, November 18, 1993)

Kun Ping LuS, Bruce E. KempQ,and AnthonyR. Means7 From the Departments of Pharmacology, Duke University Medical Center, Durham, North Carolina 27710 and the $St. Vincent's Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065,Australia

tion of the amino acid sequence of NIMA deduced from the NIMA is a cell cycle-regulated protein kinase required for theGzfM transition in the filamentousfungus Asper- nimA cDNA suggested thatthe nimAgene product belonged to gillus nidulans. Previous biochemical characterization the family of Serrrhr protein kinases. Antibodies specific for NIMA is a NIMA precipitated a fungalprotein, whichphosphorylated of the recombinant enzyme indicated that protein serindthreonine specific kinase with p-casein p-casein in vitro. Using p-casein phosphorylation, the activity being the best substrate from the many proteins and of NIMA was shown to fluctuate during the nuclear division peptides tested (Lu, K. P., Osmani,S . A,and Means,A. R. cycle, peaking in late Gz and mitosis(2). To determine the (1993)J. Biol. Chem. 268,8709-8776). However, substrate biochemical properties of the protein kinase, we expressed specificityorphysiologicallyrelevantsubstratesfor NIMA in bacteria (3). Biochemical characterization of the puNIMA remained unknown.In search for a peptide sub- rified recombinant enzyme revealed it to be a unique SerPThrstrate for this enzyme, we screened an assembled specific libraryprotein kinase, the activity of which was also regulated of synthetic peptides that each contained a phosphoby SerPThr phosphorylatioddephosphorylation (3). However, rylation site for a known protein kinase and found an excellent peptide substrate forNIMA, phospholemman nothing was known concerning NIMA substrate specificity or the identityof physiologically relevant substrates. 42-72 (PLM(42-72)). NIMA kinase phosphorylated One strategy that can be used to search for relevant subon S e P PLM(42-72)uniquelyandstoichiometrically strates of a novel protein kinase is to define the structural with aV , of 1.4 pmollmidmg and apparentK,,,of 20.0 determinants that are requiredfor phosphorylation of protein p ~ These . kinetic constants were about 10-fold higher pepand %fold lower than those for p-casein, respectively. A or peptide substrates.Of the many proteins and synthetic detailed analysis of substrate specificity determinants tides utilized as substrates for well characterized SerTIkr-spefor NIMA using synthetic peptide analogs of PLM(42-72) indicated cific protein kinases,p-casein was the best substrate (3).However, we showed that under optimal assayconditions, that Phe-ArgXaa-Serfl'hr represents the optimal primary sequence for NIMA kinase phosphorylation. Re- NIMA phosphorylated p-casein onmultiple residues and with a placement of theArg at P-2 with Ala resulted in a 6-foldrelatively low V,,, of 156 nmollmidmg (3). In this study, we V , while substi- have identified an excellentpeptide increase inK, and 2-fold decrease in substrate for NIMA, NIMA phos- namely phospholemman 42-72 (PLMl(42-7211, by screening an tution of the Phe at P-3 with Ala abolished phorylation. These results reveal the unique nature of assembled library of 56 synthetic peptides representing the substrate recognition by the NIMA kinase and should phosphorylation sites in manydifferent proteins. NIMA kinase prove valuable in the search for biologically relevant and a phosphorylated PLM(42-72) with a 10-fold higher V,, NIMA substrates. lower K, than p-casein. After establishing that the unique residue phosphorylated by NIMA in this peptide (SeF3) was different from those phosphorylated by cyclic AMP-dependent The NIMA kinase is the product of a cell cycle regulatory protein kinase (PKA) and protein kinase C (PKC), we synthegene that was isolated by genetic complementation of a tem- sized two series of peptide analogs to define the structural perature-sensitive mutation in the n i m A gene of Aspergillus determinants requiredfor NIMA kinase specificity. Our results nidulans (1). Cells carrying temperature-sensitive mutations reveal hitherto unknown unique sequence determinants rein then i m A gene werespecifically arrested inGz at the restric- quired for phosphorylation of synthetic peptide substrates by tive temperature, but rapidly and synchronously entered mito- the NIMA kinase. These results also demonstrate the utility of sis when shifted to the permissive temperature. In contrast, using a synthetic peptide screen to identify efficient substrates overexpression of the n i m A gene product induced premature for new protein kinases and provide essential information to mitotic arrest (1).These results indicate that NIMA plays a begin the search for the biological substrates for NIMA. critical role in the progression of cells into mitosis. ExaminaEXPERIMENTALPROCEDURES * This work was supported by Research Grant GM-33976 from the Synthesis of Peptides-Peptides were synthesized and purified as National Institutes of Health (to A. R. M.) and grants from the Anti- described previously(4).All peptides were dissolved in distilled water at Cancer Council of Victoria and the National Health and Medical Re- a concentration of 4 mg/ml and a series of dilutions was made using 0.25 search Council (toB. E. K.). The costs of publication of this article were mg/ml bovine serum albuminas a carrier. The precise concentration of defrayed in part by the payment of page charges. This article must each was determined by amino acid analysis, which also estabtherefore be hereby marked "aduertisement" in accordancewith 18 lishedpeptide the predicted amino acid composition of all the synthesized pepU.S.C. Section 1734 solely to indicate this fact. t Present address: Molecular Biology and Virology Laboratory, The tides. Salk Inst. for Biological Studies, P. 0. Box, 85800, San Diego, CA921865800.

ll To whom correspondence should be addressed: Dept.of Pharmacology, DukeUniversityMedicalCenter, P. 0. Box 3813, Durham, NC 27710. Tel.: 919-681-6209; Fax: 919-681-8461.

Theabbreviations used are: PLM,phospholemman;PKA, cyclic AMP-dependent protein kinase; PKC, protein kinase C, HPLC, high performance liquid chromatography.

6603

NIMA Kinase Specificity

6604

TMLEI Phosphorylation of synthetic peptides by NIMA kinase Peptides were assayed in duplicate reactions at a concentration of 0.4 pg/pl. Level of phosphorylation is expressed as percentage of the &casein phosphorylation seen in parallel assays using 1.5 pg/pl. Serial no.

Peptide name

Peptide sequence

Relative phosphorylation %

61 50 40 43 42 41 36 46 47 48 30 28 57 60 51 19 32 4 21 1 7 8 9 11 12 22 23 25 62 13 10 5 26 55 54 56 44 45 14 37 35 52 53 17 24 18 33 34 31 38 3 20 29 58 59 2

Casein Phospholemman(43-71) Phospholamban(1-31) Phospholamban(8-21) ADR1(222-234)L227 ADR1(222-234)R232 ADR1(222-234)G233 ADRl(222-234) ADR1(222-234)FZ8 ADR1(222-234)SZ3l ADR1(222-234)1232 ADRl(225-241) PTHrP(-13 to +2)C-13 DARP-32(2242)RZ9 HMG-CoA reductase(861-876) CaMKII(297-310) GS(1-10)R3A9A10desT5 GS(1-12)A9JoK"~'2 GS(1-10)T7A9A'0 PKC(19-31)S35 MLC(l1-23) MLC(l1-23)A" MLC(ll-23)Al2 MLC(ll-23)AlGR15 MLC(ll-23)A13 MLC(ll-23)Al4.l5 MLC(ll-23)Gls MLC(ll-23)Vls MLC(ll-23)G15L18 MLC(ll-23)W14

CKFNQQQRTGEPDEEEGTFRSSIRRLSTRRR MDKVQYLTRSAIRKASTIEMPQQARQNLQNL TRSAIRRASTIEMP LKKLTLRASFSAQ LKKLTRRASFRAQ LKKLTRRASFSGQ LKKLTRRASFSAQ LKKLTRKASFSAQ LKKLTRRASSSAQ LKKLTRRASFIAQ LTRRASFSAQSASSYAL CRSVEGLSRRLKRAV PRQVEMIRRRPTPAMLFRLS HLVKSHMIHNRSKINL RRKLKGSILTTMLA PLRRLSVAA PLSRTLSVAAKK PLSRTLTVAA RFARKGSLRQKNV KKRPQRATSNVFA AKRPQRATSNVFA KARPQRATSNVFA KKRPRAATSNVFA KKAPQRATSNVFA KKRAARATSNVFA KKRAARAGSNVFA KKRAARAVSNVFA KKRAGRALSNVFA KKRWQRATSNVFA KKRAARATXNVFA MLCK(11-23)A14A'5cis-hydroxy-P19 csMLC(1-17)A2" PAAAKRRAAEGSSNVRS csMLC(5-17) KRRAAEGSSNVRS MLCK(38-511 RRKLKGAIKTTKLA r-A~etyl-CoA-carboxylase(71-85)R~~~~" RRHMRSAMAGLHLVK GP120 consensus(304-338)V3loop CTRPNNTRKSIHIGPGRAFYTTGEIIGDIRQAHC mWA(14-32)AI9 QNGGVAVSYLYFSRIRRCS HCT'QYIRHGSKPMYT Phe hydroxylase(263-277) cGPKb(72-83) PRTKRQAISAEP K4PHOS.1 KKKKQISVRGLG ~GPK(77434)S~~ LRQSFRKFT VIF(181-193)C'8' CKGHRGSHTMNGH cGPK(55-67) PRTTRAQGISAEP C G P K ( ~ ~ - ~ ~ ) A ~ ~ S ~ ~ PRTARAQSISAEP S6(229-239)A235 AKRRRLASLRA S6(229-239)A23G AKRRRLSALRA S6(229-239)A238 AKRRRLSSLAA S6 kinase(723-733) RRVKKLPSTTL S6 kina~e(723-733)A~~O RRVKKLPATTL IP3 kinase(2-16) ARPRGAGPCSPGLER IP3 kinase(l98-210) KRSSEPEHYCLVR J B analog PTHRP(1-34) AITAEQAFHDKGKTVQSMNRINFLKRIVSDLRSA Tyr hydrox(2-19) PTPSAPSPQPKGFRRAVA RDYLIDGSRGIL Vlnculin(105-126) A CoA-carboxylase chick(79-98) MSGLHLVKQGRDRKKVDVQR Phos B kinase A(1014-1023) FRRLSISTES P-hydroxlase(l0-19) VLSRKLSDFG

Peptide Phosphorylation-NIMA protein kinase was purified from a bacterial expression system as described previously (3). Purified kinase was diluted prior to use in a buffer containing 50 m HEPES, pH 7.5, 0.5 mg/ml bovine serum albumin, and 1 m dithiothreitol. Phosphorylation of synthetic peptides was camed out in a 30-pl reaction mix containing 50 m HEPES, pH 7.5, 0.25 mg/ml bovine serum albumin, 10 rn magnesium acetate, 1m dithiothreitol, 100 p d or 300 PM [?"PIATP (300-1,000 countdminlpmol), and 50-100 ng of NIMA. After incubation at 30 "C for 15 min, 25-pl aliquots were removed from the reaction mix and applied to phosphocellulose P81 filters. The filters were washed with 0.5% phosphoric acid, and the radioactivity was counted as described previously (3). Under these conditions, all kinase reactions were linear for at least 30 min. Every peptide phosphorylation assay was repeated three t o six times using at least two different prepa-

100.0 1235.4 78.6 2.9 67.3 23.1 22.1 14.6 10.3 7.5 1.8 9.5 40.1 27.7 20.4 17.1 11.1 10.6 0.0 15.8 1.0 1.0 0.0 1.7 0.0 0.6 5.2 0.6 5.7 8.9 0.2 2.3

0.3 2.0 8.0 5.5 4.6 4.4 4.2 3.9 3.4 3.3 0.0 0.0 4.5 2.7 1.8 3.1 2.6 1.1 0.0 1.7 0.9 0.1 0.0 0.0 0.0

rations of NIMA kinase. Less than 10% standard deviation among experiments using different kinase preparations and less than 5% standard deviation among duplicates within a given experiment were noted. The kinetic data were analyzed as described previously ( 5 ) .Calmodulindependent protein kinase I1 and IV were assayed as described previously (6). Protein kinase A (PKA), protein kinase C (PKC), cyclin-dependent protein kinase CDC2, and casein kinase I and I1 (Upstate B~otechnologyInc.) were assayed according to themanufacturer's eonditions. Mitogen-activated protein kinase was kindly provided by Dr. Perry Blackshear (Dept. of Biochemistry,Duke University Medical Center) and assayed as described (7). Analysis of Phosphorylated Residues-For phosphoamino acid and phosphopeptide analyses, peptides were phosphorylated in thepresence of 50 p,t [f'z-PIATP. Phosphopeptides were separated from the free

6605

NIMA Kinase Specificity radioactive ATP onan AGl-X8 ion-exchange column inthe presence of A 30% acetic acid as described previously (8). After repeated lyophilization in water, peptides were subjected to partial acid hydrolysis for phosphoamino acid analysis or repeated trypsin digestionfor phosphopeptide analysis, followed by two-dimensional separation by thin layer chromatography as described previously (9). For phosphopeptide sequencing, peptides were phosphorylatedthe in B presence of 500 unlabeledATPcontaining a trace amount of [?"PIATP.Thephosphopeptideswere separated from unphosphorylated peptides by reversed-phase HPLC usinga gradient of 2041% acetonitrile (v/v) in 0.1% trifluoroacetic acid (v/v) with a flow rate of 1mumin. The purified phosphopeptides were digested overnight at 37 "C in 100 pl of 0.1 M NH4HC03 containing about 2% (w/w) protease V8 (Boehringer Mannheim). Following lyophilization, peptides were again separated by reversed-phaseHPLCusing 520% acetonitrile (v/v). The single radioactive peak was sequenced by Dr.RichardCook(Baylor College of Medicine) usingan Applied Biosystems Inc. 470A gas-phase sequenator and a 120A phenylthiohydantoin aminoacid analyzer. RESULTS AND DISCUSSION Screening the Assembled Peptide Library-To identify peptide substratesof the NIMA kinase, we used 56 syntheticpeptides representing sites identified in a wide range of proteins that are phosphorylated by many different proteinkinases (10). As shown in Table I, dramaticdifferences in theNIMA activity toward thesepeptides wereobserved. Most of the peptides were not phosphorylated by NIMA. Only eight peptides were phosphorylated a t a rate above 20% of that observed using p-casein as a substrate. However, a singlepeptide PLM(42-72) was observed to be a better substrate than p-casein. NIMA phosphorylated this peptide at a rate that was more than 10-fold faster than p-casein. Identification of NZMA, PKA, and PKC Phosphorylation Sites on PLM(42-72)"When the peptide PLM(42-72) was incubated with NIMA for 1 h, about 0.8 mol of phosphate was incorporated per mol of the peptide, suggesting thatPLM(42-72) was phosphorylated on a single residue.To identify the NIMA phosphorylation site on PLM(42-72), phosphoamino acid analysis, phosphopeptide isolation, and sequencingwere carried out. Phosphoamino acid analysis indicated that NIMA exclusively phosphorylated a serine residue (Fig. 1B 1. There are3 serines in thepeptide PLM(42-72) at positions 62,63, and 68 (Fig. lA). To distinguishwhether NIMA phosphorylated Ser62-Ser63 a n d o r S e P , phosphopeptide analysis was undertaken. The phosphorylated synthetic peptide was cleaved exhaustively with trypsin, and thedigest was separatedby thin layer chromatography according to theprotocol of Boyle et al. (9).A single phosphorylated peptide, peptide 1(Fig. E ) , was detected, indicating thatNIMA could phosphorylate either Ser62-Ser63or S e P , but not both, since trypsin cleaved a t arginines located between these serines. To identity the phosphorylated residue(s), peptide PLM(42-72) was phosphorylated by NIMA in the presence of ATP containing a trace amount of [+'z-P]ATP, the phosphorylated product was purified by reversed-phase HPLC, and digested repeatedly with proteaseV8 under conditions where the protease cleaves peptides specifically at the COOH-terminalside of Glu residues (Fig. lA). Subsequent separation by HPLC yielded a single radioactive peptide (data not shown). This peptide was subjected to amino acid sequencing. The sequence obtained was GTFRSS*IRRLSTRRR, where * indicates that thedetection of SeF3 wasselectively reduced, compared to the other residues. These results indicated that Ser63 was most likely the unique residue phosphorylated by NIMA. Sincephospholemman was previously shown to be phosphorylated by both PKA and PKC (111, we examined whether peptide PLM(42-72) was also a substrate for these protein kinases and if so, which residue(s) was phosphorylated. PKA and PKC each phosphorylatedthe peptide exclusively on serine

v8

PKC

PKA

NIMA

Y

C

NIMA

NlUArPKA

*!?

"2

PKA

NIYA+PKC

PKA+PKC

4

1

.2

c i

L ILC

hc. 1. Determination of residues in PLM(42-'72) phosphorylated by three differentprotein kinases. A, the predicted proteolytic products generated by digestion with either trypsin (solid arrows) or V8 protease (open arrows)which cleaves the peptide specifically at the COOH-terminal side of Arg or Glu residues, respectively. B,phosphoamino acid analysis. PLM(42-72) was phosphorylated by the different protein kinases as indicated and phosphorylatedpeptideswere isolated and subjected to acid hydrolysis, followed by two-dimensional

separation on thin layer chromatography plates. S,phosphoserine; T, phosphothreonine; Y , phosphotyrosine. C, phosphopeptideanalysis. The phosphorylated peptides generated by the enzymes shown at the top of each panel were exhaustively digested with trypsin and then subjected to two-dimensional separation on thin layer chromatography plates separately (left panel) or in combination (right panel ). The ratios of phosphopeptide radioactivity loaded on the thin layer chromatography plates containing the combined samples were 3:l for N I W K A , N I W K C , and PKAPKC. residues, as did NIMA (Fig. 1B). Phosphopeptide analysis indicated that a single tryptic peptide, peptide 2, was phosphorylated by PKA and that themobility of this peptide was different from that phosphorylated by NIMA, as determined by mixing experiments (Fig. E ) . These results indicate that PKA predominantly phosphorylated Ser68, which contains the appropriate consensussequence RRXS for phosphorylation by PKA. Two tryptic peptides(phosphopeptides 1 and 2) were phosphorylated by PKC a t a ratio of approximately 4:l (Fig. 1 0 . In phosphopeptide-mixing experiments, these two peptides comigrated with peptides 1and 2 which were independently phosphorylated by NIMA and PKA, respectively. Given the specificity requirements of PKC, it seems reasonablethat it phosphorylates SeP3 (peptide 1)and S e P (peptide 2) where peptide 2 contains themost favored site which corresponds to

NIMA Kinase Specificity

6606

TABLEI1 Kinetics of phosphorylation of phospholemman peptides by NIMA Peptide phosphorylation was determined as described under “Experimental Procedures.” Kinetic constants were estimated as previously described (5). Peptide

PLM(42-72) PLM(43-72) PLM(49-72) PLM(5P72) PLM(58-72) PLM(60-72) PLM(65-72) PLM(58-70) PLM(58-70) PLM(58-67)

,,V

K“t

Sequence

CKFNQQQRTGEPDEEEGTFRSSIRRLSTRRR KFNQQQRTGEPDEEEGTFRSSIRRLSTRRR RTGEPDEEEGTFRSSIRRLSTRRR 1.4 DEEEGTFRSSIRRLSTRRR 1.5 GTFRSSIRRLSTRRR FRSSIRRLSTRRR IRRLSTRRR GTFRSSIRRLSTR 1.3 GTFRSSIRRLST 1.1GTFRSSIRRL

80

Vm,lKm

PM

prnol/rnin/mg

x 10-3

20.0 17.5 19.0 20.3 44.6 110.6

1.4 1.4

71

76 1.6 0.9 Negligible 1.7

81.6 85.3 68.5

75 36 8

20 16 17

TABLEI11 The role of specific amino acids in peptide phosphorylationby NIMA Kinetic constants were determined as described under “Experimental Procedures.” Peptide

PLM(58-72) SIRRLSTRRR 38 PLM(58-72)A68.69 PLM(58-72)A62,68.69 SIRRLAARRR PLM(58-72)A61*68.69 SIRRLAARRR 4 PLM(58-72)A65,68,69 PLM(58-72)A66.68.69 SIRALAARRR PLM(58-72)A68.69,70 PLM(58-72)A60,68,69 PLM(58-72)A63 AIRRLSTRRR

K“.

Vmax

VmdL

W 44.8 94.9 70.8 260.1 165.7 152.0 106.0

prnol/rnin/mg

x 103

1.9

21 27

Sequence

GTFRS GTFRS GTFRA GTFAS GTFRS GTFRS GTFRS GTARS GTFRS

1.7 SIRRLAARRR 2.0 1.1 SIARLAARRR 1.4 1.5 SIRRLAAARR 1.7 SIRRLAARRR

the sequence RWZSXR previously foundt o be a strongly recognizedmotif in peptide substrates. Thus although peptide 2 contains sites of phosphorylation for both PKA and PKC, it seems likely that they phosphorylate SeP8 and SeP9, respectively. PKA appears to suppress PKC phosphorylation of SeF3 (peptide l ) ,but this has not been quantified (Fig. 1C).A more detailed comparison between PKC and PKA phosphorylation preferences is beyond the scope of this study. Effect of Peptide Length on Kinetics of PhosphorylationSince the NIMA phosphorylation site in PLM(42-72) was different from the major phosphorylation sites for PKA and PKC, we wished to define the determinantsfor substrate recognition by the NIMA kinase. Our strategy was to first determine the minimal peptide that could still be phosphorylated byNIMA with reasonable K,,, and V,,, values. Once this was determined, we could then introduce individual amino acid replacements with confidence that interpretation of results would be independent of variation of peptide length. We first synthesized a series of peptides with different numbers of residues deleted from the N H 2 and/or COOH terminus of the parent peptide and examined them as substratesfor NIMA (Table 11).The removal of residues from the NH2 terminus up to Pro53 didnot affect the kinetics of peptide phosphorylation. Although omitting Asp54, Glu, Glu, Glu57slightly increased the apparent K , (2-fold),it did not alter the V,,,. Further deletion of GlP8 and ThP9 substantial19 reduced the V,,, for peptide phosphorylation and also increased the apparent K,, suggesting that the glycine and threonine residues contribute to some extent to the affinity of the peptide for the kinase. As expected, negligible phosphorylation was observed with the peptide in which the phosphorylation site, SeF3, was deleted (PLM-65-72). Deletions up to S e P residue from the COOH-terminal side had only a small effect on the kinetics, i.e. all peptides were phosphorylated at rates comparable to those observed with the peptide, PLM(5872). These results indicated that the COOH-terminal residues up to S e P do not play a major rolein substrate recognition by the NIMA kinase.

9 10 16 Negligible Negligible

Phosphorylation of Peptides in Which Individual Residue(s) Are Changed to Alanine-% identify the amino-terminal specificity determinants in substratesfor NIMA kinase, we investigated the role of individual residues in the minimal peptide substrate PLM(58-72). A series ofPLM(58-72) analogs was synthesized in which a single or multiple residues were substituted with alanines. As mentioned above, this approach allowed the identification of the functionally important amino acids independent of the potentially confounding effectsof peptide length variation. The kinetic constants for phosphorylation of these peptides by NIMA are shown in Table 111. Substitution of Ser63with Ala gave a peptide that was not phosphorylated by NIMA, while substitution of SeP8 and ThP9 or Ser6’, Ser68, and ThP9 with alanines had littleeffect onthe overall kinetics of peptide phosphorylation by NIMA. These results confirmed that NIMAphosphorylated a single residue, SeP3. WhenAre5, or Arg70were replaced singly or collectively with Ala, the kinetic constants were onlyslightly altered. However, replacing with Ala increased the apparent K , about 6-fold and also reduced V,,, resulting in a much poorerpeptide substrate and indicated that the Arg at position -2 from the phosphorylated Ser(P-2) may be important for substrate recognition. The most striking finding of these studies was that the substitution of complete inhibition of peptide PheG0with Ala resulted in a near phosphorylation by NIMA. This indicates that a Phe at P-3 is absolutely required for substrate phosphorylation and may represent a distinguishing feature of NIMA substrate recognition when compared to other known protein kinases. In support of this unique requirement for NIMA substrates, we compared phosphorylation of two peptide substrates of NIMA as potential substrates for several other Ser/Thr protein kinases. As shown in Table IV, only NIMA and PKCcould phosphorylate PLM(58-72)A62,68,69. Substitution of PheG0with Ala (PLM(58-72)A60,68,69) reduced peptide phosphorylation by NIMA approximately 20-fold whereas this change had only a modest but opposite effecton phosphorylation by PKC increasing it by 1.5-fold. These data reveal that even though NIMA

Are6, Are1

NIMA Kinase Specificity

6607

TABLE IV Phosphorylation of selected PLM peptides by different proteinkinases Peptide phosphorylationwas determined as described under “Experimental Procedures.” The amounts (ng) of kinases used are: NIMA, 20; PKC, 5; PKA, 280; ( M A P ) kinase, 200; casein kinase I, 250; casein kinase 11,25; calmodulin (CaM)-dependentkinase 11,217; CaM-dependent kinase IV, 259, and cyclin-dependent CDC2,250. ~~~~~~~

Protein kinase

Phosphorylation

58GTFRASIRRLAARRR72 (PLM(58-72) A62,68,69)

NIMA PKC PKA MAP kinase Casein kinase I Casein kinase I1 CaM-dependent kinase I1 CaM-dependent IV Cyclin-dependent CDC2

100.0 * 1.9 60.9 4.4 9.9 1.9 1.0 * 0.2