Biochimica et ~iophysica Acta, 1094 (1991) 67-76

67

© 1991 Elsevier Science Publishers B.V. 0167-4889/91/$03.50 ADONIS 016748899100215W

BBAMCR 12972

Minireview

Intrasteric regulation of protein kinases and phosphatases B r u c e E. K e m p a n d R i c h a r d B. P e a r s o n St. Vincent's Institute of Medical Research, Fitzroy, Victoria (Australia) (Received 30 March 1991)

Key words: Intrasteric control; Protein kinase; Phosphatase; Inhibitory site; Latent form

Contents I.

Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

67

II.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

67

IlL

Location of pseudosubstrate regions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

68

IV. Myosin light chain kinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

72

V.

73

Protein kinase C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

VI. Calcineurin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

73

VII. Protein phosphatase-1 inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

74

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

75

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

75

1. Summary Protein kinases and protein phosphatases are the pre-eminent regulators of cellular processes. Many of these enzymes are present in latent forms that are activated by various modulators. The inhibited form is maintained by autoinhibitory domains either within these proteins or in some instances by separate inhibitory subunits. A number of these autoinhibitory structures have been identified because of structural similarity to their enzyme's substrate. These findings indicate that the enzyme's active site may recognize either substrates or pseudosubstrate autoinhibitory

Correspondence: B. Kemp, St. Vincent's Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065, Aus:ralia. Abbreviations: PKI, protein kinase inhibitor, PKC, protein kinase C

structures that turn them off. Because this form of regulation is directed at the active site it is termed intrasteric control.

11. Introduction In many cases the functions of protein kinases and phosphatases are latent and require the dissociation of inhibitory subunits or autoinhibitory regions to express enzyme activity. Removal of these regulatory regions occurs in response to an activator binding, or covalent modification such as phosphorylation or dephosphorylation. For the cAMP-dependent protein kinase [1] and protein phosphatase-1 [2], dissociation of a regulatory subunit or specific inhibitor protein is required for activation, the subunit inhibits enzyme activity by mimicking a substrate and binding in the active site. In

68 o t ~ : ~nstances, such as myosin light chain kinase [3] e~d ~:~rotein kinase C [4], an internal region of the enzyme is responsible for autoinhibition. Again this autoinhibitory region resembles the substrate, binds to the active site and thereby denies the substrate access. The binding of the allosteric activators results in conformafional changes: that remove the pseudosubstrate ~gion from the active site. ~ u s , pseudosubstrate regulatory regions may be present on separate subunits, inhibitory proteins or be contiguous with the sequence of the core catalytic domain (Fig. 1). At present the definition of a pseudosubstrate is rather loose, including any internal structure that folds back on the active site to turn the enzyme off or an external structure contributed by an inhibitory subunit. in both cases, the regulation of enzyme activity involves an autoinhibitory region that exploits the specificity and structure of the active site. In order to highlight this form of regulation, we have termed it intrasteric control, in contrast to allostedc control where the regulators bind at distinct sites and tend not to be structurally related to the suhstrate [5]. It seems likely that at the molecular level the pseudosubstrate/active site interactions will not precisely mimic those of the substrate/active site in much the same way that antiidiotype/antibody interactions for lysozyme do not mimic all of the lysozyme/antibody interactions at the molecular level [6]. Intrasterically regulated enzymes may be monomeric or multimeric, existing in homomeric or heteromeric forms (Table I). Intrasteric and allosteric regulation represent different links in the same chain of events with aUosteric control being required to modulate the intrasteric interactions. For example, the phorbol ester binding site in the zinc finger motif of protein kinase C

A l t o a ~ Site X e Z u ~ nonu~

Active Site

~

~

Pscadom~um ~i

Fig. 1. Model of intrasteric regulationof enzymesby a pseudosubstrate reidon contiguous with the catalytic domain. The enzyme is rnaintainuJ in an inactive state due to binding of the pseudosubstrate region, masking the active site. Allosteric activation results in the release of the pseudosubstrate reSioo following binding of an activator or covalent modification.

[7] is adjacent to the pseudosubstrate region exerting the intrasteric control (see below). The concept of intrasteric control has been developed largely for protein kinases and to a limited extent protein phosphatases, however it is likely to occur for other enzymes including the aromatic hydrozylases [8,9] or fibroblast collagenase [10] whose activities can be substantially enhanced by proteolysis. It is not yet clear whether intrasteric regulation can be reasonably extended to encompass phosphorylation of protein kinases within their core catalytic domain such as occurs for cdc-2 [11] and a number of protein tyrosine kinases [12] where the internal phosphate group may bind to substrate recognition residues in the catalytic core and block substrate access.

IlL Location of pseudosubstrate regions The recognition of protein kinase pseudosubstrate structures has depended very much on our knowledge of the substrate specificity of protein kinases as well as the determination of their primary structures and the proximity of aUosteric regulator sites (see below). The pseudosuhstrate sequences identified for a number of protein kinases are listed in Table II. At present it is not known what proportion of protein kinases are regulated by pseudosubstrate structures. In most cases described so far the pseudosubstrate sequence is contiguous with the amino acid sequence of the catalytic domain and acts intramolecularly, although the cAMP-dependent protein kinase contains is pseudosubstrate sequence on a separate regulatory subunit (Table I). All protein kinase pseudosubstrate sequences identified so far are located outside the core catalytic domain. Identification of Arg as a key specificity determinant for the cAMP-dependent protein kinase [13] and the synthesis of peptide substrates and inhibitors [14] paved the way for understanding the mechanism of inhibition of the cAMP-dependent protein kinase by the regulatory subunit [15]. Historically, the pseudosubstrate structure in the regulatory subunit was referred to as the 'hinge region' [1] because of its particular sensitivity to proteolytic cleavage. There is now compelling evidence from proteolysis studies, chemical modification and site directed mutagenesis that supports the pseudosubstrate regulatory concept for the cAMP-dependent protein kinase (reviewed in Ref. 1). The cAMP-dependent protein kinase inhibitor protein (PKI) also contains a pseudosubstrate domain [16,17]. Recognition o'f a protein kinase's substrate specificity determinants is not necessarily straight-forward, there is considerable variability in phosphorylation site sequences even for protein kinases whose substrate specificity has been investigated exhaustively [18]. This variability adds to the uncertainty in identifying pseu-

69 TABLE I

lntrastericaily regulated protein kinases and phosphatases Enzyme Monomeric Protein kinase C Substrate Pseudosubstrate

Substrate/pseudosubstrate sequence

Activator

Phorbol ester

AKRRRLSS*LRA RI9FARKGA*LRQKNV31

smMLCK Substrate Pseudosubstrate

1SSKRAKAKTTKKRPQRATS*NV 78TSKDRMKKYHARRKWQKTGHA*VR

Calcineurin (phosphatase) Substrate Pseudosubstrate

DLDVPIP6RFDRRVS(P)*VA ITSFEEAKGLDRINERMPRRDA*RP

Homomeric Cam I! PK (a 2) Substrate Pseudosubstrate cGMP-PK (a 2) Substrate Pseudosubstrate Heteromeric cAMP-PK (R 2C2) Substrate Pseudosubstrate regulatory subunit RI regulatory subunit RII PKI Phos. b kinase (a/33,/~)4 Substrate Pseudosubstrate a 3' subunit Protein phosphatase-1 Substrate Pseudosubstrate Inhibitor-1 a Inhibitor-2 site a a Inhibitor-2 site b a

Calmodulin/Ca2+

Calmodulin/Ca2+

Calmodulin/Ca2+ PLRRTLS*VAA flZSlHRaETVDCLKKFNARRKLKGA*ILR cGMP RKRS*RAE GPRTT*RAQG*ISA cAMP LRRAS*LP

RRRRGA*IS RRVS*V¢ RRGA*IS Calmodulin/Ca2+ KRKeIS*VRGL RDPYA*LRPL

DQEKRKeIS(p)VRG EQI RRRRPT*P 36 EREKKRQFE*NKR142 ELSKKSQKWDE*RNIsl

cAMP-PK GSK-3

a Hypothetical pseudosubstrate site. See Tables !! and IV for references. GSK-3, glycogen synthase kinase-3; PKI, cAMP-dependent protein kinase inhibitor. * Phosphate acceptor site or residue thought to ocupy the phosphate acceptor site.

dosubstrate structures. Nevertheless, as the mechanisms operating for different classes of protein kinases are determined, it will become increasingly feasible to recognize the pseudosubstrate motifs of related family members by inspecting the amino acid sequence alone, as has been done for protein kinase C [4]. In Table II, a number of pseudosubstrate sequences are shown and these indicate some of the common features that may be used as a guide in identifying pseudosubstrate regions. Ala is the most common residue occupying the phosphate acceptor site but Gly, Thr and Ser can also occur. The frequent use of Ala is not surprising given its structural similarity to Ser. The residue in the phosphate acceptor site is frequently

followed by a hydrophobic residue (Val, lie or Leu). The presence of the Ala-hydrophobic residue can be critical for the potency of pseudosubstrate peptide inhibitors. For example, substitution of I1e-22 with Gly in the cAMP-dependent protein kinase inhibitor peptide PKI (5-22), T5TYADFIASGRTGRRNA122 decreases potency more than 100-fold [17]. On the other hand, proteolysis and mutation studies with the smMLCK [19,21] indicate that neither Ala-806, that occupies the phosphate acceptor site, nor Val-807 are required for inhibition of smMLCK activity. These studies imply that not all of the smMLCK pseudosubstrate sequence (STSTKDRMKKYMARRKWQK TGHAV s°7) is required for intrasteric regulation in

7O TABLE 1I Protein kinase autoregulatory pseudosubstrate sequences Protein kinase Protein kinase-Cfamily (19-31) I and I1 (19-31) 3' (16-30) Drosophila (26-40) • (151-165) A(152-166) 8 (139-153) (111-125) yeast (393-407) Calmodulin-dependentprotein kinases Cam-PK It family a (280-307) t. (281-308) 1' (281-308) a (281-308) Cam PK.Gr (53-80) b Calspermin (1-24) Myosin light chain kinase family Fibroblast (1072-1095) Smooth muscle (787-810)

Skeletal muscle(570-593) twitchin(5 416-5 439) Phosphorylasekinase 3' subunit Rabbit (332-353) c

Sequence

Reference

ANRFARKGA*LRQKNV TVRFARKGA*LRQKNV RPLFCRKGA*LRQKVV MKSRLRKGA*LKKNVF MRPKKRQ6A*VRRRVH HFTRKRQRA*mRRRVH FPTNNRRGA*IKQAKI AESIYRRGA*RRgRKL HGGLHRHG&*IINRKE

52 53 54 55 56 57 58 58 59

CNHRQET*VDCLKKFNARRKLKGA*ILTTM NNHRQET*VECLKKFNARRKLKGA*ILTTN MHHRQET*VECLKKFNARRKLKGA*ZLTTN HNHRQET*VDCLKKFNARRKLKGA*ILTTN NFVHHDTAQKKQEFNARRKLKAA*VKAVV MDTAQKKQEFNARRKLKAA*VKAVV

60 61 62 62 63 64

SKDRMKKYMARkKWQKTGHA*VRAI SKDRMKKYMARRKWQKTGHA*VRAI SeRLLKKYLHKRRgKKNFIA*VSAA SQIPSSRYTKIRDSZKTKYDA*MPE

32 3 65 66

VIRDPYA*LRPLRRLIDAYAFRI

67

cAMP-dependent protein kinase regulatorysubunits cAMP-PKR I subunit (92-107) (bovine, haman, pig) RRRRGA*ISAEV (mouse) RRRRGG*VSAEV ( Diclyostelium) RKRRGA*ISSEP

1 68 69

cAMP-PKR il subunit (97-105) (bovine, pig, rat) (yeast, 147-155)

RRV$*VCAET RRTS*VSGET

1 70

Protein kinase inhibitor(5-26)

TTYADFIASGRTGRRNA*IHDIL

17

cGMP-dependent protein kinase cGMP-PK a (54-69) cGMP-PK ~ (70-83) ¢ Drosophilo-Gl gene (137-150) -G2-TI gene (474-485)

6PRTT*RAQG*ISAEP SEPRT*KRQA*ZSAEP NPAAIKKQG*VSAES qNFRQRALG*ISAEP

71

72 73 73

" Thr-286 is autophospborylatedhoweverthe GAIL sequence resembles other pseudosubstrate sequences closely. b Thr-59 the equivalent to Thr-286 does not have a nearby Arg comparable to Arg-283. c Hypotheticalassignment. * Phospborylated residue or residue thought to occupy the phosphate accepter site.

situ. However, the full length pseudosubstrate peptide is required for optimum potency in vitro. Phosphorylation sites often occur near or at the proposed pseudosubstrate regulatory site. These ind u d e Ser-95 for the RII subunit [1] of the cAMP-dependent protein kinase, Thr-58 [1] for the cGMP-dependent protein kinase a form, Thr-286 [22] for the calmodulin-dependent protein kinase II and Ser-16 and Thr-17 adjacent to the pseudosubstrate region of t h e / 3 form of protein kinase C (PK C) (19-31) [23].

The presence of autophosphorylation sites in or near the proposed pseudosubstrate regulatory regions poses the question of their significance and whether a phosphorylatable residue lies in the active site in the latent form of the enzyme. For the cAMP-dependent protein kinase regulatory subunit, RII Ser-95 [1] probably does occupy the active site and the phosphorylated regulatory subunit does inhibit the catalytic subunit, albeit with reduced affinity [24]. For protein kinase C the autophosphorylation sites are clearly distinct from the

71 pseudosubstrate site [23] and no single site is phosphorylated stoichiometrically. Although Thr-286 autophosphorylation is important in rendering the calmodulindependent protein kinase II independent of calmodulin it is not clear that this occupies the active site in the autoinhibited form of the enzyme. Indeed, inspection of the pseudosubstrate sequences in Table II suggests that the GAIL sequence fits more closely the features of a pseudosubstrate site. This is analogous to protein kinase C where the autophosphorylation sites occur upstream of the pseudosubstrate site. Nevertheless, the results obtained with synthetic peptides indicate that synthetic peptides Thr-286 are the most potent inhibitors of calmodulin-dependent protein kinase II [22]. Autophosphorylation sites provide information on the proximity of various regions to the active site. Knowing the position of autophosphorylation sites may well provide a guide to locating regulatory sequences, particularly when little is known about the enzyme's substrate specificity. It seems reasonable to expect that some protein kinases that are regulated by protein phosphorylation may turn out to have regulatory phosphorylation sites adjacent to pseudosubstrate regions. Once a putative pseudosubstrate sequence has been located outside the catalytic domain of the protein kinase in question, synthetic peptides corresponding to this region can be tested as inhibitors. While synthetic pseudosubstrate peptide inhibitors can be very potent (K i = 2-10 nM) this is not always the case as the corresponding synthetic peptides of the pseudosubstrate regions oi' the cGMP-dependent and cAMP-dependent protein kinases are poor inhibitors (Table III) [25]. Several experimental approaches can be used to investigate whether the identified region is responsible for intrasteric regulation of the protein kinase. These can include the use of proteolytic fragments of the enzyme with the pseudosubstrate region removed to render it constitutively active. Alternatively, the pseudosubstrate region can be removed by truncating the enzyme with site directed mutagenesis. Another approach is to study the effect of polyclonal antibodies

directed at the pseudosubstrate region on enzyme activity. Protein kinase C was completely activated by an antibody to the pseudosubstrate region [26]. We have not yet been successful in applying this strategy to activate smMLCK, indicating that it may not be a universal method. Raising antibodies to regions adjacent to putative pseudosubstrate sequences may well be a useful alternative way of activating protein kinases, analogous to binding an allosteric activator to an adjacent site. Technical difficulties will vary with each protein kinase and restrict some approaches; for instance recombinant protein kinase C devoid of the pseudosubstrate region is very unstable and cannot be isolated readily [27]. Although there is compelling evidence that pseudosubstrate regions can inhibit protein kinases and that this most likely occurs by binding via the active site, the available data fall short of formal proof. Current X-ray crystallographic studies by Taylor and her colleagues with the cAMP-dependent protein kinase inhibitor complex are expected to resolve some of these questions. The identification of pseudosubstrate structures within protein phosphatases is less advanced. The lack of detailed knowledge of the substrate specificity determinants of these enzymes has made recognition of potential pseudosubstrate structures difficult. An autoinhibitory domain has been reported for the calmodulin-dependent protein phosphatase calcineurin (also termed protein phosphase 2B) based on characterizing constitutively active forms of the enzyme activated by proteolysis and testing peptide analogs of the potential autoinhibitory region for the ability to inhibit activity [28]. This autoinhibitory domain contained a region homologous to the phosphorylation sites of several of calcineurin's substrates and a synthetic peptide analogue of this region inhibited calcineurin activity. The protein phosphatase-1, inhibitors 1 and 2 may also act via pseudosubstrate structures (see below). The following examples detail how the pseudosubstrate regions have been identified for the myosin light chain kinase, protein kinase C, calcineurin and protein

TABLE 11I

Protein kinase synthetic pseudosubstrate inhibitor peptides

Protein kinase

Sequence

Ki/IC5o (/~M)

Reference

cAMP-PK cGMP-PK ( - form) (/3 form) Protein Kinase C Cam-II PK

TTYAD F I~tSGRTGRRNA*IHD PRTTRAQ6*|SAEP PRTKRQA*ISAEP RFARKGA*LRQKNV PlHRQET*VDCLKK FNARRKLKGA*I LTTPILA LKKFNARRKLKGA*I LTTMLA

0.003 2100 900 0.13 2.7 24

17 a a 4 74 75

0.012

36

smMLCK(787-807)

SKDRIqKKYIqARRKWQKTGHA*V

a Michell,B. Robinson, P.J. Mitchelhill,K.I. Kemp, B.E. (1991) unpublisheddata.

72 phosphatase-1. The regulation of other enzymes including the cAMP-dependent protein kinase [1], cGMP-dependent protein kinase [1] and calmodulindependent protein kinase II [22,29] have been reviewed recently and will not be discussed further here. IV. Myosin fight chain kinase The myosin light chain kinase (MLCK) is regulated by calcium and calmodulin. Its mechanism of activation by these regulators has been studied in several laboratories [3,19,21,30-33]. The gizzard smMLCK pseudosubstrate structure was recognized because of the similar juxtaposition of basic residues in the calmodulinbinding domain to that of the local phosphorylation site sequence in the myosin light chains [3] (Table I). The domain structure of smMLCK is shown in Fig. 2 with the regulatory domain containing the overlapping pseudosubstrate and calmodulin-binding domains on the carboxyl side of the catalytic domain. Three approaches have been used to investigate the regulation of smMLCK including synthetic peptides [3], proteolytic fragments [19,30,33] and recombinant enzyme [21,32]. It was found that synthetic peptides corresponding to this region were potent substrate antagonists [3,20] consistent with this region acting as an inhibitor. Structure/function studies on the pseudosubstrate sequence have been undertaken in several laboratories [3,34-36]. The potency of the corresponding synthetic peptides depends on the full length of the pseudosubstrate sequence. The second approach involved the use of proteolysed forms of the smMLCK [19,33]. A calmodulin-independent 61 kDa tryptic fragment of the enzyme devoid of its pseudosubstrate region can be generated (Fig. 2). An intermediate 64 kDa inactive tryptic fragment is also produced that results from cleavage at Art-808 in the calmodulinbinding domain. This fragment does not bind caimodulin, but since it retains the pseudosubstrate sequence it remains inactive. On the other hand, endoproteinase 147

240

288

481

526

Lys-C cleaves smMLCK at Lys-802 to generate an inactive fragment [20] indicating that it is not necessary for all of the pseudosubstrate sequence to be present to inhibit the enzyme. The sequence TS°3GHAV s°7 is therefore not essential in the enzyme, but is required for optimum potency of the pseudosubstrate inhibitor peptide in vitro. Addition of a large excess of substrate peptide does not compete with the endogenous pseudosubstrate and activate smMLCK, again emphasising possible differences between the binding of peptide substrates and the endogenous pseudosubstrate. The results obtained with the proteolytic fragment of smMLCK are consistent with the recent results obtained with recombinant smMLCK, truncated in the pseudosubstrate region. Ito et al. [21] found that truncation at Lys-793 near the amino terminal end of the pseudosubstrate region (Ser-787) resulted in a constitutively active fragment, but truncation at Trp-800 resuited in a fragment that was largely inactive. These results suggest that the core autoinhibitory region is the sequence Y~94MARRKW s°°, located in the pseudosubstrate region, consistent with it being responsible for inhibition of the smMLCK. Proteolysis experiments have also been undertaken with the skMLCK [31], however these have provided equivocal results due to the instability of the proteolytic fragment containing the pseudosubstrate sequence that becomes com~itutively active during isolation. In contrast to this consistent body of data supporting the pseudosubstrate hypothesis for smMLCK regulation there are two reports that have questioned its interpretation. Ikebe et al. [30] reported that the 61 kDa constitutively active tryptic fragment of smMLCK terminated at Lys-776, while the 64 kDa inactive tryptic fragment had a ragged end terminating at Lys-793 and Art.797. This data suggested that the inhibitory domain encompassed K~NMEAKKLS78~KDRMKK ~93. Their results are inconsistent with both our own proteolysis studies and the recombinant protein studies of Ito et al. [21]. Moreover, the synthetic peptide, 762

787

815

857

931 972

VKKPAPKT2s~PPK

K rrtNMEAKKLSKDRMKKYMARRKWQKTGHAVRsos

61 kDa active fragment

]

[64 kDa inactivefragment Fig. 2. Schematic representation of the domain structure of protein kinase C. The relative positions of the pseudosubstrate and phorbo] ester binding site to the catalytic domain are shown.

73 D777TKNMEAKKLSKDRMKK 793 containing this region was more than three orders of magnitude less potent than the pseudosubstrate peptide, MLCK 787807 [20]. Shoemaker et al. [32] reported a series of mutations in the pseudosubstrate calmodulin-binding region of fibroblast MLCK. Significantly, substitution of the adjacent basic residues R R K 799 (residue numbering as for smMLCK) with EEE did not result in a constitutively active enzyme although substitution of each of 6 basic residues between Lys-784 and Lys-793 with Glu did result in constitutively active enzyme. One interpretation is that this region acts as a 'hinge' and that charge mutations in this region are more effective at removing the pseudosubstrate sequence from the active site than mutations in the three adjacent basic residues RRK 799. Whatever the molecular basis, these observations illustrate the limitations of the techniques used and emphasize the need for three-dimensional structural information. V. Protein kinase C

The identification of the pseudosubstrate autoregulator in protein kinase C was made by inspecting the amino acid sequence for structures that resembled the best synthetic peptide substrates known at that time, ribosomal protein $6 (229-239) AKRRRLSS* LRA and EGF receptor (650-658) VRKRT*LRRL [37]. The pseudosubstrate sequence identified was RFARKGALRQKNVHEVKN corresponding to protein kinase C/3 (19-36) [38]. It is located adjacent to the phorbol ester binding domain (Fig. 3). Certair, protein kinase C isoenzyme forms e ,~ ~5 sr and yeast) have large extensions on the amino terminal side of the pseudosubstrate sequence (Table II). It should be recognized that there is great diversity in the local phosphorylation site sequences for protein kinase C [18] that could have obscured identification of the pseudosubstrate sequence had all these possibilities been taken into account. Obtaining direct evidence to support this model of regulation for protein kinase C has not been straightforward. While the corresponding synthetic peptides were potent inhibitors [4,38] it was difficult to undertake mutation studies of the pseudosubstrate rePseudosubstrate

",,

PhorbolEster

/

BindingSRe

REGULATORYDOMAIN

CATALYTICDOMAIN

Fig. 3. Schematic representation of the domain structure of smMLCK. The structure of the catalytic and regulatory domains of smMLCK

shown together with the sites cleaved by trypsin that generate the inactive 64 kDa fragment and the constitutively active 61 kDa

are

fragment.

gion because of instability of the mutant enzyme [27]. Using co-transfeetion of protein kinase C with the pseudosubstrate region deleted and a phorbol ester reporter gene it was possible to show that it was constitutively active [27]. Structure/function studies of the pseudosubstrate peptide have been undertaken and these have shown that Arg-22 is critically important, because substitution of this residue with Ala results in a 600-fold reduction in potency [38]. The pseudosubstrate peptides have been particularly useful reagents in exploring the function of protein kinase C in a number of systems [39-42]. Indeed, the expressed E isoenzyme of protein kinase C does not phosphorylate histone, but was found to phosphorylate the Ser analogue of its pseudosubstrate peptide [43]. This was the first example of the use of a pseudosubstrate structure to design a substrate for a protein kinase that had never been isolated, but whose eDNA structure was known. Although there are minor differences in the pseudosubstrate sequences for the protein kinase C isoenzyrnes (see Table II), the corresponding peptides do not show isoenzyme specificity [44]. VL Ca|cineurin

Caicineurin is the brain isoenzyme of protein phosphatase 2B, the calcium/calmodulin-dependent phosphatase. Proteolysis of calcineurin was found to generate a constitutively active form of the phosphatase analogous to the 61 kDa fragment of smMLCK, implying the removal of an autoinhibitory region [45]. The autoinhibitory region was mapped to the carboxyl terminal 40 residues, some 50 residues from the putative calmodulin-binding domain. This contrasts with the smMLCK and calmodulin-dependent kinase II where the autoinhibitory and calmodulin-binding domains overlap. Hashimoto et al. [28] further localised the autoinhibitory domain of ealcineurin by using a series of peptide analogues of the carboxyl terminus and testing their ability to inhibit dephosphorylation of skeletal muscle myosin light chains at limiting concentration. The peptide, I476TSFEEAKGLDRINER491M PPRRDAMP 5°°, inhibited calcineurin with an IC50 = 10 ~M. Inhibitor activity was abolished by removal of the sequence M492pPRRDAMP s°° which resembles the phosphorylation site sequences of several of calcineurin's substrates. The amino terminal residues, I476TSFEEAKGLDRINER 49s, in the inhibitory peptide appear to be important for inhibition because the extended carboxyl terminal peptide, P4~RRDAMps°° SDANLNSINKALASETNGTDSNGSNSSNIQ 5s° ineluding this potential pseudosubstrate site did not inhibit calcineurin activity. A similar influence of amino terminal residues has also been reported for a calcineurin synthetic substrate, where it was found that addition of the sequence DLDV to the peptide PIP-

74 GRFDRRVS(P)VAAE, greatly enhanced the kinetics of dephosphorylation [46]. This peptide is an analogue of the phosphorylation site in the R-If regulatory subunit of cAMP-dependent protein ldnase. The calcineurin autoinhibitory region contains analogous acidic regions with RII and Other calcineurin substrates, but their role in the mechanism of inhibition is not known. VII. Protein phosphatase-I inhibitors Two heat-stable inhibitors of protein phosphatase-1 (inhibitor-1 and inhibitor-2) have been purified and characterised [2]. Inhibitor-I activity requires phosphorylation of Thr-35 by cAMP-dependent protein kinase in the sequence L2~DPEAAEQIRRRRPT(P)PATLV 4°. This sequence shares multiple basic residues and a conserved acidic residue with the phosphorylation site in phosphorylase a (Table IV). Foulkes et al. [47] previously proposed that inhibitor-1 binds to phosphatase-1 at a site enabling access of the phosphorylated Thr-35 to the active site, The dephosphorylation of Thr-35 in inhibitor-I by phosphatase-1 is competitively inhibited by inhibitor-2 provided Mn 2+ is present, Thus, under these conditions, it is clear that Thr-35 does occupy the active site of phosphatase-1. The specific requirement for Mn 2÷ may not be unexpected because chemical studies have shown that the phosphothreonine at residue 35 with adjacent Pro residues is remarkably stable [48]. Inhibitor-1 has been reported to give mLxed competitive inhibition with phosphorylase a as substrate [47], but ~his may not be especially significant in terms of it acting as pseudosubstrate because complicated kinetics are also obtained with protein kinase pseudosubstrates peptides [22,33,35]. Inhibitor-1 has a K m of 190 nM as a substrate for phosphatase-1, but a K t as an inhibitor of between 1.5 and 7.5 nM [47], but this is analogous to smMLCK where the K m for peptide substrate phosphorylation is 200-fold greater than the K t for the pseudosubstrate peptide analogue. Proteolytic fragments of inhibitor-1 have been tested and 9-54 is active but not 13-40 [49]. Thus, if inhibitor-1 is acting

as a pseudosubstrate, then the potency of inhibition is dependent on a rather long peptide, analogous to the smMLCK pseudosubstrate peptide. Because proteolytic fragments of the inhibitors have been used in these structure/function studies, high concentrations of the peptides have not been available to assess weak inhibitory activity (micromolar to mUlimolar range) analogous to the cAMP-dependent protein kinase regulatory subunit RII pseudosnbstrate peptide. Additional synthetic peptide and recombinant protein studies are required to address these questions. The mechanism of action of inhibitor-2 is also complicated, since at low concentrations (K D -- 0.1 nM) its inhibition of the phosphatase-1 catalytic subunit can be reversed by glycogen synthase kinase-3 phosphorylation at Thr-72 but at higher concentrations (K D ~- 5 nM) its actions are not reversed by phosphorylation [50]. Nonetheless, inhibitor-2 does inhibit phosphatase 1 competitively using both phosphorylase a and inhibitor-1 as substrates [47]. Inspection of the phosphatase inhibitor-2 sequence reveals two hypothetical pseudosubstrate sequences shown in Table IV. The sequence EREKKRQFE* M 14° exhibits a similar juxtaposition of basic residues and conserved Glu to the putative pseudosubstrate site in inhibitor-1. In this region the sequence FE*M 14° would be expected to occupy the active site. A related sequence KKSQKWDEM is present in the amino terminal region of inhibitor-2 with a similar E*M 49 and adjacent basic residues on the amino terminal side. This site is just upstream from the Thr-72 site that is phosphorylated by glycogen synthase kinase-3. The proteolytic fragment 24-114 had only 2% of the inhibitor-2 activity, suggesting that higher orders of structure may be important in this instance [51]. The presence of multiple basic residues and Met in the putative pseudosubstrate sequences provides an explanation for the sensitivity of the phosphatase inhibitor to digestion with cyanogen bromide and trypsin. However, considerable further direct evidence is required to substantiate that these regions of inhibitor-1 and inhibitor-2 do in fact act as pseudosubstrate inhibitors of phosphatase-1.

TABLE IV

Hypothetical psetedombstrate regions in phosphata~e inhibitors:l and -2 Protein

%quence

Reference

Substrate phesphorylase b ]lthibitor-1 Inhibitor-2 site a Inbibitoro2 site b

DQEKRKQ ZS * V RS K91qFTVPLLEPHLDPEAAEQIRRRRPT,I, PAT LV LTS OQSSPEVEDR ;5,; R l l ] I REQESSGEEDSOLSPEEREKKRQFE*HF-.R~ LHYNEGLNIKLARQL T159 Sa3RVASAE(IPRGSVDEELSKKSQKWDE*MN i LATYHPADKDYGLM (KZ) DEPST.PYHSHZZ

76 49 49

• , indicates the location o f the phosphate aceep~or site or pseudosubstrate.

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