Communication Vol. No. THE OF CHEMWY The American Society … · 2001-06-30 · Communication Vol. 269, No. 47, Issue of November 25, pp. 29343-29346, 1994 0 1994 by The American

Communication Vol. 269, No. 47, Issue of November 25, pp. 29343-29346, 1994 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Printed in U.S.A.

THE JOURNAL OF BIOLOGICAL C H E M W Y

Mammalian 5’-AMP-activated Protein Kinase Non-catalytic Subunits Are Homologs of Proteins That Interact with Yeast Snfl Protein Kinase*

(Received for publication, September 15, 1994, and in revised form, October 4, 1994)

David StapletonS, Guang GaoQS, Belinda J. Michell, Jane WidmerQ, Ken Mitchelhill, Trazel Teh, Colin M. House, Lee A. WittersQ, and Bruce E. Kempl From St. Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065, Australia and the $Endocrine-Metabolism Division, Dartmouth Medical School, Hanover, New Hampshire 03755-3833

The B’-AMP-activated protein kinase is responsible for the regulation of fatty acid synthesis by phosphoryla- tion and inactivation of acetyl-coA carboxylase. The porcine liver 5”AMP-activated protein kinase 63-kDa catalytic subunit co-purifies 14,000-fold with a 38- and 40-kDa protein (Mitchelhill, K. I. et aZ. (1994) J. BioZ. Chern. 269, 2361-2364). The 63-kDa subunit is homolo- gous to the Saccharomyces cerevisiae Snf l protein ki- nase, which regulates gene expression during glucose derepression. Peptide amino acid and polymerase chain reaction-derived partial cDNA sequences of both the pig and rat liver enzymes show that the 38-kDa protein is homologous to Snf4p (CAT3) and that the 40-kDa protein is homologous to the Siplp/Spm/GAL83 family of Snflp interacting proteins. Sucrose density gradient and cross-linking experiments with purified B’-AMP-acti- vated protein kinase suggest that both the 38- and 40- kDa proteins associate tightly with the 63-kDa catalytic polypeptide in either a heterotrimeric complex or in di- meric complexes. The 40-kDa subunit is autophosphoryl- ated within the 63-kDa subunit complex. The sequence relationships between the mammalian 5’-AMP-activated protein kinase and yeast Snflp extend to the subunit proteins consistent with conservation of the functional roles of these polypeptides in cellular regulation by this family of metabolite-sensing protein kinases.

The 5’-AMP-activated protein kinase phosphorylates and in- hibits the rate-limiting enzymes in the fatty acid and sterol synthesis pathways (acetyl-coA carboxylase and HMG-CoA re- ductase, respectively). This regulation, as well as the observa-

* This work was supported by the National Heart Foundation (Aus- tralia) and the National Health and Medical Research Council (to B. E. K.) and by National Institutes of Health Grant DK35712 (to L. A. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The protein sequence(& reported in this paper has been submitted to the EMBL Data Bank with accession number(s) P80385-P80387.

ll National Health and Medical Research Council Fellow. To whom $ These authors contributed equally to this work.

4162676. correspondence should be addressed. “el.: 61-3-2882480; Fax: 61-3-

tion that honnone-sensitive lipase is also a substrate for the kinase, has led to the concept that this kinase plays a coordi- nating role in the control of lipid metabolism (reviewed in Ref. 1). The regulation of lipid metabolism by the 5’-AMP-activated protein kinase is most significant under conditions of cellular stress, such as metabolic fuel limitation, ATP depletion, and heat shock (2-6). The phosphorylation of hydroxymethylglu- taryl-CoA reductase and acetyl-coA carboxylase has been rec- ognized for several years (1,7,8). Recently, sufficient quantities of purified porcine Ei‘-AMP-activated protein kinase were ob- tained to provide partial amino acid sequence information (9) that revealed a striking amino acid sequence homology be- tween the 63-kDa subunit of the porcine 5‘-AMP-activated pro- tein kinase and the yeast Snfl protein kinase subfamily (9). The complete cDNA sequence of a rat homolog of the porcine 5‘-AMP-activated protein kinase, which also is homologous to Snflp, has recently been reported (10). The 5”AMP-activated protein kinase and Snfl protein kinase share functional prop- erties; both kinases phosphorylated and inactivated yeast acetyl-CoAcarboxylase (9). Additional support for this relation- ship has now been provided by Wood et al. (111, who found that glucose limitation in Saccharomyces cerevisiae was accompa- nied by parallel activation of Snflp and inactivation of yeast acetyl-CoAcarboxylase. This finding in yeast also parallels pre- vious observations in mammalian cells, where glucose deple- tion leads to apparent activation of the B’-AMP-activated pro- tein kinase (5).

Snfl protein kinase was initially identified because it was required for the expression of glucose derepressible genes (12) and interacts with a number of proteins. Snf4p (CAT3)’ is physically associated with Snflp (9,13-15) and co-purifies with it (see below). Using the two hybrid system, Carlson and her colleagues (16,17) have identified other Snflp interacting pro- teins including Siplp, Sip2p (also referred to as Spm2p), and sip3 (16, 17). Siplp is a substrate for the Snfl protein kinase in vitro (16).

In view of the close structural and functional relationship between the 5’-AMP-activated protein kinase and Snfl protein kinase, it was of interest to investigate whether the 38- and 40-kDa proteins that copurified with the 63-kDa catalytic sub- unit of the 5’-AMP-activated protein kinase were related to these Snflp-interacting proteins. In the present study, we re- port that, based on both peptide and PCR2-derived cDNA se- quences of pig and rat liver enzymes, the 38-kDa protein is a mammalian homolog of Snf4p (CAT31 and the 40-kDa subunit is a homolog of Siplp.

EXPERIMENTAL PROCEDURES Enzyme Purification-The 5‘-AMP-activated protein kinase was pu-

rified from porcine liver and rat liver as described previously (9). Peptide Sequencing-The 5’-AMP-activated protein kinase prepara-

tions were run on SDS-PAGE (7.5-15% gradient gel) that had been polymerized for at least 8 h. Gels were stained with Coomassie Blue, destained in acetic acid/methanoYwater (7/12.5/81.5, v/v), and then washed extensively in water. Bands for sequencing were excised from the gel and processed as described previously (9). Either Asp-N endo- proteinase or tryptic peptides were purified with a Brownlee RP-300 C8

CAT3 is the formal name (13); SNF4 is also widely used. Nomen- clature use is e.g. Snflp is the product of the SNFI gene.

PAGE, polyacrylamide gel electrophoresis; BS3, bis(sulfosuccinimidy1) ‘The abbreviations used are: PCR, polymerase chain reaction;

suberate.

29343

29344 AMP-activated Protein Kinase Subunit Structure

reversed phase column (2.1 x 250 mm) using a Hewlett-Packard 1090 high pressure liquid chromatograph and developed with a standard 0.1% trifluoroacetic acid (vh) to 60% CH,CN (v/v) in 0.085% trifluoro- acetic acid gradient at 100 pVmin over 60 min. Peaks were monitored at 214 nm and collected manually. Each peak was rechromatographed on a Hypersil ODS column (5 pm, 2.0 x 100 mm), and the peaks were N-terminally sequenced on either an Applied Biosystems 471A protein sequencer or a Hewlett-Packard GlOOOA protein sequencer.

PCR-derived cDNAs for 38- and 40-kDa Subunits-Peptide se- quences for the 38- and 40-kDa proteins (see Figs. 2u and 3) were used to generate a series of degenerate oligonucleotides for reverse tran- scriptase-PCR. In brief, cDNA was synthesized from either pig or rat liver total RNA using either oligo-dT,, or peptide-derived degenerate primers and reverse transcriptase. The cDNA was then amplified with selected degenerate primers (and nested primers), chosen by prelimi- nary partial alignment of peptide sequences with the homologous yeast proteins (see "Results"). The major products were separated on ethidium bromide-agarose gels, the bands cut out, glass wool purified, and cloned using either TA cloning (Novagen) or PCR-Script (Strat- agene). Plasmid DNA was prepared from positive clones and sequenced using dideoxynucleotides and Sequenase (U. S. Biochemical Corp.) as recommended by the manufacturer.

Sucrose Density Gradients-Five samples were analyzed on sucrose density gradients: catalase (500 pg), bovine serum albumin (500 pg), recombinant a-casein kinase I1 subunit, casein kinase I1 (a,&), and purified 5'-AMP-activated protein kinase with or without inclusion of 100 PM 5 ' 4 " in the gradient. Each sample was made to 100 pl and applied to a 10-ml 5 2 0 % sucrose gradient in 50 mM Tris-HCI, pH 7.5, 1 mM EGTA, 1 mM EDTA, 1 mM dithiothreitol, and 300 mM NaCl, the latter to prevent casein kinase I1 from aggregating. Centrifugation was performed (100,000 xg, 19 h, 4 "C) in a Beckman SW 28 rotor. Fractions (400 pl) were collected and assayed appropriately. Catalase and bovine serum albumin were measured with the Coomassie Blue dye binding assay (18). and casein kinase I1 a-subunit, casein kinase I1 holoenzyme, and 5'-AMP-activated protein kinase were assayed in the presence of their appropriate peptide substrates (9, 19).

Protein Cross-linking-Purified 5'-AMP-activated protein kinase (1.8 pg) was incubated with increasing concentrations of the cross- linker bis(sulfosuccinimidy1)suberate (BS3) made in H,O, in a final vol- ume of 750 p1 a t room temperature for 30 min. The reactions were quenched with 100 p1 of 1 M glycine. Each sample was trichloroacetic acid-precipitated (6% v/v) and run on SDS-PAGE (7.5-17% gradient gel). Protein bands were visualized by Coomassie Blue staining.

Kinase Autophosphoylation-Prior to autophosphorylation, 14 pg of pure B'-AMP-activated protein kinase was desalted on a PD-10 gel filtration column. We observed that the phosphorylation of the 40-kDa subunit was sensitive to the salt concentration and therefore necessi- tated a desalting step. Varying concentrations of 5'-AMP-activated pro- tein kinase were incubated in a final volume of 1.4 ml in the presence of 20 [y-32PlATP (1,500 cpdpmol) for 30 min a t 37 "C. Reactions were stopped with trichloroacetic acid precipitation, and the resus- pended pellet was run on SDS-PAGE (7.5-17% gradient gel). The gel was stained with Coomassie Blue, dried, autoradiographed, and quan- titated by liquid scintillation counting. Results were plotted as activity per mass of 5'-AMP-activated kinase protein as a function of the kinase concentration (M, = 100,000).

RESULTS AND DISCUSSION Porcine liver 5'-AMP-activated protein kinase was purified

14,000-fold using three chromatographic steps that included a peptide substrate affinity column (9). The 63-kDa catalytic sub- unit, which is related to the yeast Snfl protein kinase, co- purified with two other proteins of 38 and 40 kDa as assessed by SDS-PAGE (Fig. la). Tryptic peptide maps of each protein were different from the 63-kDa subunit, indicating that they were not partial proteolysis products of it (results not shown). Snfl protein kinase interacts with a variety of other proteins. When Snflp is purified from bakers' yeast, it co-purifies with Snf4p (CATS) (Fig. la) (9) and can be immunoprecipitated with both Snf4p (CAT31 and Siplp from yeast extracts (14, 17). We found that Siplp (M, 96,000) did not copurify with Snfl protein kinase from bakers' yeast using nickel-agarose chromatogra- phy (Fig. la) (9).

The rat and porcine 38- and 40-kDa subunits isolated by SDS-PAGE were digested with trypsin or Asp-N endoprotein-

A

AMP-activaled kinase (nM)

FIG. 1. a, SDS-PAGE of the B'-AMP-activated protein kinase and yeast Snfl protein kinase. The gel was run as described under "Exper- imental Procedures." A parallel sample of the 5'-AMP-activated protein kinase (lane B ) was transferred to nitrocellulose, renatured, and incu- bated with [Y-~'P]ATP as described previously (12, 23); the resultant autoradiograph is shown. Only the 63-kDa band and not the 38- or 40-kDa bands autophosphorylated following renaturation. Snf4p copu- rifies with Snflp (lane E) . The minor bands running under Snflp are proteolytic fragments of Snflp determined by protein sequencing. A protein of approximate M, >200,000 is present in variable amounts between 5'-AMP-activated protein kinase preparations (see, for ex- ample, Fig. 4b where i t was not evident). Blotting with streptavidin- peroxidase or enzyme-specific antibodies indicates the presence of acetyl-coA carboxylase (Gao et al., unpublished observations). Protein sequencing of peptides derived from this region of the gel gave se- quences identical to non-muscle myosin. b, the effect of dilution on

bated at varying dilutions in the presence of [y-32PlATP as described autophosphorylation. The 5'-AMP-activated protein kinase was incu-

under "Experimental Procedures." The autophosphorylated enzyme was analyzed on SDS-PAGE and autoradiographed. Radioactivity in- corporated into the 63-kDa (0) and 40-kDa (0) subunits was quanti- tated by liquid scintillation counting.

ase (9) and the resultant peptides sequenced. Using degenerate oligonucleotides and PCR, we also cloned partial length cDNAs for both the pig and rat 40-kDa proteins (Fig. 2a). These pep- tide and cDNA sequences reveal that the 40-kDa protein is a mammalian homolog of the S. cereuisiae proteins GAL83 (Spmlp (417 amino acids)), Sip2p (Spm2p (415 amino acids) and Siplp (863 amino acids) (Fig. 2b). The protein sequence, 124 residues, obtained for the 40-kDa subunit showed 20.2% identity with Siplp, previously identified as a substrate for Snflp using the two-hybrid system (161, 27.4% identity with Sip2p (Spm2p), and 22.6% identity with GAL83. Both Siplp and Sip2p belong to the Gm83 family of transcriptional regu- latory proteins required for expression of GAL genes (20). Pre- vious comparisons of the GAL83 sequence with both Siplp and Sip2p revealed that the homology was largely confined to two regions, one in the center and one at the C terminus (20). The cDNA sequence of the 40-kDa subunit spans the center seg- ment and contains 13 completely conserved residues between the 40-kDa subunit, GAL83, SipBp, and Siplp (see Fig. 2b). In addition there are 13 identical residues conserved between

AMP-activated Protein Kinase Subunit Structure 29345

liver 40-kDa subunit sequences. Peptide FIG. 2. a , comparison of rat and pig

sequences were obtained as described un- der “Experimental Procedures.” The pep- tide sequences were aligned according to the cDNA-derived sequence. Peptide amino acid (aa) sequences are under- lined. Phenylthiohydantoin-amino acids with equivocal identification are notated X . The shaded residues in the partial cD- NAs are identical between pig and rat cDNA and/or peptide sequences. b, com- parison of 40-kDa subunit, GAL83, Sip2p, and Siplp. Shown is the alignment of the rat 40-kDa protein to Sip2p (123-2491, GAL83 (120-2471, and Siplp (446-622). The sequences, aligned with the Pileup program (GCG, University of Wisconsin), were formatted by an Excel macro with the residues identical to the 40-kDa pro- tein sequence shaded (22).

a R~ICDNA n . -.-I F Q T ~ M 4 - 4 n E W P - . ,““,‘,-“-~~~a.A.~U~ ~ a t a a I I M D S P E D A D I F H T E E I K E E F L A W Q H D I E V N E K Rat aa Plg cDNA Plgaa I I M D S P E D A D L Y H S E E I K E E F L A X Q H D I E V N D K

P T V F R E V Y L S G S F ~

A P E K

r n l kB O m s - - r - ” W ~ - - . ~ ~ ~

Plg aa D L Y H S E E I K A P E A S A Q A R P T V F R X T G G G K E V Y L S G S F

RstcDNA . - a . - . F , . .- - I Ralaa N N W X X I P I T R Rat aa Plg CDNA Plgaa N N S S K

F F V D G Q W T H D P S E P I V T S Q L G T V N N I I Q V K K T D S Q N N F V A I L D L P E G E H Q Y K

Plg aa I P L T R X H N N F V A I I D I P E G F L V D G Q X T H D P S E P V V T S Q D P S E P V V T S Q I G T V N N I I Q V K K X X

b R a l 4 0 k c D N A m M D ~ E ~ A D I F ~ T E E M K A P E K E E F ~ ~ ~ D ElpZ(spm2) K Q D A D l D R S G S S P R E E G Q Q Q I R A K E A S I E V N ~ A P O Q A R [ T ~ F ~ ~ ~ ~ G G P S I K S S L M V ’ V E I

Slpl m T N G L N L Q P L H P L H P 1 I N D N E S Q Y S O P h R E I S H H S N S M S S M S S I S S T N S T E N T I V U L K K D D

Rat 40k cDNA . .

GAL^^ N T L Q K M D Y Q P S Q Q P D S L Q N ~ G F Q Q Q Q E Q , Q G . T V E G K G R ~ M M F - V D I T

Rel4OkcDNA[;; 51p2(5pm2) ; l ; R Q W T H D n N E L R V S D F L ’ T A P S E l l V l S [ L l T D.M N F V V “ 1 1 Y I E I a[ K K T D

GAL83 R F R I B N E L R F S D Y I - T A D.M N F V Y M E R O P E S A P P

SIP1 Y R L Q S I N I I T H S N F L - T A D S E N F V W F E L P G Y H T I E P F R N E A

GAL83, SipBp, and Siplp that are not conserved in the 40-kDa subunit. It seems probable that the 90-residue region contain- ing the invariant residues will represent a conserved structural motif common to this family of proteins. The PCR-derived cDNA sequence permitted us to align the peptide amino acid sequences that covered essentially its entire length and pro- vided compelling evidence that the cDNA sequence is correct and not that of an isoform or other member of the GAL83 family (Fig. 2a). The cDNA-derived sequences of the pig and rat forms of the 40-kDa fragment are highly homologous with 94.3% identity, indicating high sequence conservation between mam- malian species.

The peptide and corresponding partial length PCR-derived sequence for the 38-kDa subunit revealed that it was related to Snf4p (CATS) with 34.7% identity over 285 residues (Fig. 3). Comparison of the rat and pig amino acid sequences obtained from peptide sequencing indicated that they were highly con- served across species with 104/105 residues identical.

Renaturation experiments confirmed that the 63-kDa sub- unit of 5‘-AMP-activated protein kinase autophosphorylates (Fig. la) as has been shown for Snfl protein kinase (12). Pre- viously Carling et al. (21) reported that partially purified rat liver 5’-AMP-activated protein kinase was autophosphorylated on a 63-kDa band, and this polypeptide could be affinity-la- beled with [14C]fluorosulfonylbenzyl adenosine, an ATP analog. The 38- and 40-kDa proteins show no autophosphorylation ac- tivity following renaturation after electrophoresis (Fig. la). However, prior incubation of native 5’-AMP-activated protein kinase in vitro with [y-32PlATP resulted in phosphorylation of both the 63-kDa subunit and the 40-kDa protein (Fig. l b ) . Phosphorylation of the 40-kDa protein was greater than that of the 63-kDa subunit; from two-dimensional gel electrophoresis, it was apparent that there are multiple phosphorylation sites (possibly three or four, results not shown). Phosphorylation of both the 63-kDa subunit and the 40-kDa protein was independ- ent of dilution over the range 2-16 nmol of the 63-kDa subunit (Fig. lb) . These results suggest that 40-kDa autophosphoryla- tion occurs by an intramolecular mechanism catalyzed by the 63-kDa subunit within the enzyme complex.

Sucrose density gradient centrifugation of the 5”AMP-acti- vated protein kinase revealed that it sedimented with an ap- parent molecular mass of 103 k 1.5 kDa (Fig. 4a). The presence ofAMP did not alter the of the 5’-AMP-activated protein kinase sedimentation pattern, indicating that it did not activate the enzyme by causing dissociation of the complex. The M , of 103,000 estimated by sucrose density gradient centrifugation is comparable with the earlier estimate of the molecular mass of

100 2 30 kDa reported for the rat enzyme using gel chroma- tography on Superose-12 (21). Addition of the masses of the three 5’-AMP-activated protein kinase subunits determined by SDS-PAGE in the presence of reducing agent gives a total of 141 kDa, substantially higher than the value for a complex obtained from sucrose density gradient centrifugation. The ap- parent mass of the 40-kDa protein obtained from SDS-PAGE is 37 kDa when run in the absence of reducing agent (data not shown); there is still not good agreement between the two methods of estimation of the complex mass. If the values ob- tained by SDS-PAGE are overestimates, then there is a hetero- trimeric kinase complex consisting of the 63-, 40-, and 38-kDa proteins. Alternatively, the 5’-AMP-activated protein kinase complex could consist of a nearly equal mixture of dimers of 63- and 40-kDa subunits together with dimers of 63- and 38-kDa subunits, but we consider this less likely.

Further evidence in favor of the concept that all three pro- teins are part of a complex (or complexes) was obtained by cross-linking experiments, where it was found that increasing concentration of cross-linking agent BS3 caused the disappear- ance of all three proteins and the appearance of an approximate 200-kDa species (Fig. 46). At intermediate concentrations of cross-linker, a 120-kDa species was apparent but disappeared at higher concentrations of the cross-linker. These cross-linking experiments were done at low protein concentrations to favor intramolecular cross-linking. We were unable to use sedimen- tation equilibrium analysis because attempts to concentrate the purified 5”AMP-activated protein kinase (in 2 M NaC1,30% ethylene glycol-containing buffer) and transfer it into a suitable buffer were not successful with the amounts of enzyme avail- able. Whether heterotrimer or mixtures of heterodimers, it seems reasonable to conclude that the 40- and 38-kDa proteins can be regarded as kinase subunits. Recently the rat liver en- zyme has been purified by ATP-y-Sepharose chromatography (22), and the 63-kDa catalytic subunit was reported to copurify with 38- and 35-kDa proteins consistent with earlier observa- tions with purified porcine liver 5’-AMP-activated protein ki- nase (9). Using glycerol density gradients the mass of the rat liver enzyme was estimated to be 190 f 10 kDa (22). While somewhat higher than their earlier estimate of 100 kDa (21) it is nevertheless apparent that the enzyme most likely exists as a heterotrimer although a mixture of heterodimers cannot be unequivocally excluded. As a point of nomenclature, we propose that the catalytic subunit (63 kDa) be designated the a subunit, the 40-kDa protein the p subunit, and the 38-kDa protein the y subunit of the mammalian 5’-AMP-activated protein kinase.

29346 AMP-activated Protein Kinase Subunit Structure S F 4 U I P I O D S O E ~ V S I E O O L * V E S I R I F L N S r T S l D Y L P Y S Y R R 1 1 3 g l m w aDU Rat ea

FIG. 3. Comparison of 38-kDa sub- rwWA ; w!; unit sequence with Snf4p. Shown is apu

the alignment of Snf4p and the rat partial cDNA-derived 38-kDa subunit sequence rmmw with the aligned peptide amino acid (aa) w- sequence from both pig and rat enzymes. 2:: Residues identical to the 38-kDa cDNA- 9~. T X H D K S T ~ M O N I R K R

derived sequence are shaded. Homology :?- between the 38-kDa Drotein and S n f 4 ~ ex- 2:

C L I M L E S R S ( I

. . . . .

I L W E S I E A I I N R L V E ! E “ k :

tends over the entire length of Snf4p. . v - Pa, u

kDa

200

116 97

66

45

31

0 20 30 60 80 1 0 0

BOTTOM Gradient Volume (a) TOP

0.0 0.1 0.2 0.5 1.0 2.0 5.0 10.0 BS3 (mM)

FIG. 4. a, sucrose density gradient centrifugation of the 5’-AMP-acti- vated protein kinase. The porcine enzyme was centrifuged as described under “Experimental Procedures” with (0) or without (0) AMP present in the gradient. The positions of the markers, casein kinase a2P2 (130 kDa, 0) and casein kinase a subunit (40 kDa, A) are as indicated. Not shown for clarity are the positions of bovine serum albumin and cata- lase markers. The apparent M, values were calculated as described by Martin and Ames (24). 6 , cross-linking of 5’-AMP-activated protein kinase. Purified porcine 5’-AMP-activated protein kinase was cross- linked with varying concentrations of BS3 for 30 min as indicated and analyzed by SDS-PAGE. The disappearance of the 63-, 40-, and 38-kDa subunits was accompanied by the transient appearance of a 120-kDa diffuse band followed by a 200-kDa diffuse band.

Given the homologies between the piglrat and yeast proteins, it is tempting to speculate on the roles of the mammalian 38- and 40-kDa kinase subunits. Cross-complementation between GAL83 and Siplp and Sip2p in yeast suggests that all three proteins have similar functions (20) and raises the possibility that the 5’-AMP-activated protein kinase, through its 40-kDa subunit, may regulate transcription. Since GAL83, SIPl, and

SIP2 null mutations or any pairwise combinations do not affect regulation of the GAL1 gene, it has been suggested that the three genes are redundant and that additional members of the family remain to be identified (20). I t seems reasonable that there will be corresponding multiple counterparts in mamma- lian cells, but they may not necessarily all associate with pro- tein kinases. Active SnflpISnf4p protein kinase complex iso- lated from bakers’yeast does not contain detectable amounts of a 96-kDa protein (Fig. la) corresponding to Siplp, indicating that Siplp is not essential for enzyme activity (9), nor does the SnflplSnf4p complex co-purify with any other proteins in stoi- chiometric amounts. However, our purification strategy for SnflplSnf4p relies on nickel-agarose binding to the polyhisti- dine tail of Snflp, so that any proteins associating with Snflp via this region may not be recovered. I t seems reasonable that the 40-kDa subunit of the 5‘-AMP-activated protein kinase may not be essential for enzyme activity, although it is clearly tightly bound. Although the 40-kDa subunit is a substrate for the 63-kDa catalytic subunit of the 5’-AMP-activated protein kinase complex i t can nevertheless be purified by the peptide substrate affinity column in the presence of 500 mM NaCl (9).

In summary, this study has shown that mammalian liver cells contain a family of proteins involved in the regulation of lipid metabolism that are homologous to the Snflp, Snf4p (CAT3), and GAL83 yeast proteins. This opens up a wide vista of opportunities for studying their structurelfunction relation- ships, physiological roles, and their genes.

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