ZIP kinase, a key regulator of myosin protein phosphatase 1

Cellular Signalling 17 (2005) 1313 – 1322 www.elsevier.com/locate/cellsig

Review

ZIP kinase, a key regulator of myosin protein phosphatase 1 Timothy A.J. Haystead* Department of Pharmacology and Cancer Biology, Duke University, Levine Science Research Building, C119, Research Drive, Durham, NC 27710, USA Received 12 April 2005; accepted 6 May 2005 Available online 11 July 2005

Abstract Two major physiological roles have been defined for zipper interacting protein kinase (ZIPK), regulation of apoptosis in non-muscle cells and regulation of Ca2+ sensitization in smooth muscle. Although much attention has focused on the role of ZIPK in the regulation of apoptotic events, its roles in smooth muscle are likely to have equal if not greater physiological relevance. We first identified ZIPK as a major protein kinase controlling the phosphorylation of myosin phosphatase (SMPP-1M) and the inhibitor protein CPI17 in smooth muscle. Phosphorylation of SMPP-1M and CPI17 by ZIPK inhibits phosphatase activity towards myosin and causes profound Ca2+ sensitization and contraction in smooth muscle. ZIPK will also directly phosphorylate both muscle and non-muscle myosin. The highly selective actions of ZIPK in the control of myosin phosphorylation potentially make the enzyme an ideal candidate for the development of novel therapeutics to treat smooth muscle related disorders such as hypertension or asthma. D 2005 Elsevier Inc. All rights reserved. Keywords: ZIP kinase; Smooth muscle myosin phosphatase; SMPP-1M; Smooth muscle; Calcium sensitization; Apoptosis; Myosin light chain; CPI17; Rhokinase; Rho A

Contents 1. Introduction . . . . . . . . . . . . . . . 2. Ca2+ sensitization and the regulation of 3. ZIP kinase . . . . . . . . . . . . . . . 4. The regulation of ZIP kinase . . . . . . 5. ZIPK as a therapeutic target . . . . . . Acknowledgements . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . .

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1. Introduction Smooth muscle plays an essential role in a wide variety of physiological processes, including the regulation of blood pressure by controlling vessel diameter in the periphery, digestive processes by controlling mechanical movement of food though the gut, reproductive functions by controlling

* Tel.: +1 919 613 8606/9; fax: +1 919 668 0977. E-mail address: [email protected]. 0898-6568/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2005.05.008

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penile erection and delivery of the fetus at birth, regulation of lung capacity through regulation of airway diameter [1]. A major factor governing the contractile state of all smooth muscles is the phosphorylation level of myosin light chain (LC20) [2]. In smooth muscle, steady state phosphorylation of LC20 is dictated by the opposing activities of myosin light chain kinase (MLCK) and myosin phosphatase (SMPP-1M) [3]. Alterations in the sensitivity of various smooth muscles to Ca2+ (Ca2+ sensitization) has been hypothesized to be an underlying cause of many diseases associated with smooth muscle dysfunction such as hyper-

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tension, bronco spasm, sexual dysfunction, gastrointestinal disorders and glaucoma. Ca2+ sensitization is observed in smooth muscles as a disproportionate increase in LC20 phosphorylation and contractile response for a given [Ca2+] [2]. Many hormone receptors acting through small G proteins (e.g. RhoA) have been shown to induce this phenomenon [2]. Therefore, understanding the signaling elements controlling myosin phosphorylation/dephosphorylation state may identify attractive points of therapeutic intervention in a variety of human diseases associated with smooth muscle dysfunction. Recently, our laboratory identified zipper interaction protein kinase (ZIPK, also known as MYPT1 kinase, ZIPlike kinase and DAPK3) as a co-localizing SMPP-1M kinase that selectively phosphorylated the regulatory subunit (MYPT1) of the phosphatase and the inhibitory protein CPI17 inducing Ca2+ sensitization in smooth muscle [4,5]. ZIPK can also directly phosphorylate both smooth muscle and non-muscle myosin regulatory light chain (LC20) of myosin [6]. Phosphorylation of LC20 in non-muscle cells is thought to be the major trigger for cellular apoptosis [7,8]. Taken together, these findings suggest that the major physiological role of ZIPK is the governance of myosin phosphorylation in both smooth muscle and non-muscle cells. We will argue that the highly selective actions of ZIPK in the control of myosin phosphorylation in smooth muscle potentially make the enzyme an ideal candidate for the development of novel therapeutics to treat smooth muscle related disorders. 2. Ca2+ sensitization and the regulation of smooth muscle myosin phosphatase 1 (SMPP-1M) Smooth muscle cells devote considerable effort to regulating the respective activities of MLCK and SMPP1M (Fig. 1 and [9 –11]. Inhibiting or activating either enzyme results in a net increase or decrease in LC20 phosphorylation and hence change the contractile state of smooth muscle. Phosphorylation of LC20 triggers a conformational change at the hinge region of myosin activating its ATPase, enabling binding to actin and force development

(contraction) [12]. Conversely, dephosphorylation of LC20 is required to reverse this process (relaxation) [13,14]. In smooth muscle, SMPP-1M is localized with myosin and shows a high degree of substrate specificity towards LC20 [15 – 17]. We and others have hypothesized that regulation of SMPP-1M activity enables exquisite control of smooth muscle contractile state independently of [Ca2+] [2,3]. Although Ca2+ is the primary signal for controlling LC20 phosphorylation through the activation of MLCK, being a small diffusible molecule, it is a somewhat crude indiscriminate signal. Activation or inhibition of SMPP-1M enables a targeted attenuation of the signal, enabling fine adjustments in the basal state (reflected by the level of LC20 phosphorylation) or more measured changes in contractile response to hormonal or neuronal stimulation. Since the activity of MLCK is largely tied to intracellular [Ca2+], alterations in the activity of SMPP-1M has profound effects on LC20 phosphorylation. Activation of SMPP-1M in response to vasodilators greatly attenuates the Ca2+ signal (Ca2+ desensitization). Conversely, inhibition of SMPP-1M greatly exacerbates the contractile effect of a given rise in intracellular [Ca2+] (Ca2+ sensitization). An extreme example of Ca2+ sensitization can be observed in isolated smooth muscles treated with the PP-1 inhibitor calyculin A, which produces a profound sustained contractile response even at very low [Ca2+] [18]. Such a high degree of control is necessary for many smooth muscles, particularly those controlling blood pressure or the airways. Large changes in the contractility of vascular smooth muscles for example would have a disastrous outcome, resulting in large unattenuated changes in blood pressure or bronco spasm in the case of airway smooth muscle [1,19]. At the same time, however, such exquisite control of smooth muscle contractility comes at a price. The molecular basis underlying many smooth muscle related disorders such as hypertension or bronco spasm may involve disregulation of the very same elements providing fine control of smooth muscle contractility. Purified SMPP-1M is a heterotrimer consisting of the 37 kDa catalytic subunit of protein phosphatase 1 (PP-1Cy), a 110 –130 kDa myosin targeting subunit (MYPT1) and a variably recovered 21 kDa protein (M21) of undetermined

Fig. 1. Current model for the regulation of myosin phosphorylation in smooth muscle by SMPP-1M.

T.A.J. Haystead / Cellular Signalling 17 (2005) 1313 – 1322

function [15 – 17]. Several splice variants of MYPT1 have been identified in different smooth muscles, which are thought to infer different contractile properties upon those tissues [20 –23]. A variety of studies by various laboratories including ours has firmly established a major role for SMPP-1M in the regulation of myosin in most types of smooth muscles as well as non-muscle cells [15 –17,24 – 27]. These studies include the demonstration that recombinant and native MYPT1 selectively binds and alters the catalytic activity of PP-1C towards myosin and not other substrates [15 –17]. When added to permeabilized precontracted smooth muscles, recombinant and native SMPP-1M relax smooth muscle [15,28]. Addition of CPI-17, a small molecular weight phosphoprotein that potently and selectively inhibits SMPP-1M activity in vitro, causes Ca2+ sensitization and contraction when added to Triton X100 skinned vascular smooth muscle [29,30]. Importantly, several studies have established that SMPP-1M activity is regulated by phosphorylation of its MYPT1 myosin targeting subunit in smooth muscles [4,31 –39]. Initial indications of regulation came from the observation that addition of GTPgS to permeabilized smooth muscles increased LC20 phosphorylation and contraction at sub-maximal [Ca2+] (Ca2+-sensitization), mimicking the actions of many agonists that exert their effects on smooth muscles through G protein coupled receptors [40 – 43]. RhoA is the major GTP-binding protein in smooth muscle [40] and this finding led to the hypothesis that the small G protein plays an essential role in the process of Ca2+ sensitization in smooth muscles by causing inhibition SMPP-1M activity (Fig. 1 and reviewed in Ref. [9]). Although several proteins have been identified as targets for RhoA [44,45], Rho-binding kinase (ROCKa and h) has emerged as the primary candidate for mediating this response by directly phosphorylating and inhibiting SMPP-1M [32 – 34,37]. Further evidence for a direct involvement of Rho kinase in the regulation of Ca2+ sensitization has come from studies with the Rho kinase inhibitor Y-27632. Treatment of isolated smooth muscles with Y-27632 selectively blocks contraction by inhibiting Ca2+ sensitization [46]. Furthermore, Y-27632 has been shown to dramatically correct hypertension in hypertensive rat models [33,46]. In vitro, Rho kinase has been shown to phosphorylate mammalian and avian forms of MYPT1 at T695/697 (chicken/rat sequence) and Ser 850/855 (chicken/rat sequence) (28 – 36). Importantly, phosphorylation T695/ 697 has been established to inhibit SMPP-1M activity towards myosin [4,31 – 39], whereas phosphorylation of Thr850/855 causes dissociation of MYPT1 from myosin [37]. Phosphorylation of T695/697 and S850/855 has been established in vivo either directly by mass spectrometry (MS) and 32P-labeling (our group) or with phosphospecific antibodies [4,32,34,37,47]. Both sites also appear to change in phosphate content in response to Ca2+ sensitizing agonists such as carbachol, GTPgS or constitutively active

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forms of RhoA when added to intact or permeabilized smooth muscles, although different laboratories report considerable variation in the extent of these changes. For example, Niiro et al. have reported T695/697 to show a high level of basal phosphorylation in a-toxin permeabilized and intact fibers with very little or no change in response to Ca2+ sensitizing agonists [48]. Changes in the level of S850/855 phosphorylation were observed in this study, along with profound changes in the phosphorylation T38 on CPI17. In contrast, we and others have shown site specific changes in Thr695/697 in smooth muscle and other cells [36,47,49]. We suspect that some of the discrepancies in MYPT1 phosphorylation state reported between laboratories is mostly attributable to inherent difficulties associated with the preparation of isolated smooth muscles from animals and the various techniques used to measure these changes. Clearly, everyone agrees that Thr695/697 and S850/855 are endogenously phosphorylated sites in smooth muscle and that alteration in their phosphate content is likely to have functional consequence for SMPP-1M activity in smooth muscle. More recently, we showed that the extent of phosphorylation of T695/T697 is also regulated by a mutual exclusion mechanism involving phosphorylation of S695/ S696 by cyclic AMP or cyclic GMP dependent protein kinases [36] (Fig. 1). Phosphorylation of MYPT1 at S695/ S696 by these kinases does not effect phosphatase activity directly, but does prevent inactivation of the phosphatase by preventing phosphorylation at T695/T697. This mechanism may provide a means of explaining the Ca2+ desensitizing and muscle relaxing effects of agonists that elevate intracellular cAMP or cGMP. Prior to our study, Surks et al. have shown that PKG directly binds MYPT1 through its C terminal leucine zipper domain, supporting a direct role for the protein kinase in the regulation of SMPP-1M in vivo [50]. Despite compelling evidence for a role for Rho kinase in mediating Ca2+ sensitization in smooth muscles, more recent studies suggest that the kinase may not directly phosphorylate MYPT1 in vivo. First, in intact muscles and other cells, RhoA is largely localized at the plasma membrane, raising both temporal and spatial concerns about the access of activated Rho kinase in relation to myosinbound MYPT1 [51,52]. Indeed, much of the evidence supporting a direct role from Rho kinase in the regulation of SMPP-1M comes largely from in vitro biochemical experiments or over-expression studies of constitutively active non-membrane bound enzyme or Rho A in cultured cells [32,43]. Second, the somewhat surprising finding that targeted disruption of Rho kinase a or h in mice does not result in an obvious loss of smooth muscle function raises further concerns over Rho kinases direct involvement in the regulation of smooth muscle contraction. Although Rho a deletion resulted in utero death in most cases, surviving null animals were viable, but no obvious smooth muscle related phenotype was reported [53]. Indeed, the reported phenotype for the surviving animals was abnormalities associated

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T.A.J. Haystead / Cellular Signalling 17 (2005) 1313 – 1322

with blood coagulation as a result of elevations in plasminogen inhibitor I levels. More recently, the same group of investigators reported targeted deletion of Rho kinase h [54]. Rho kinase h null mice were also viable, but specifically exhibited abnormal eyelid and ventral body closure. Examination of epithelial cells of the eyelid did show disorganization of filamentous actin that was also associated with a loss of LC20 phosphorylation, supporting a Rho kinase involvement. Beyond this observation in eyelid epithelial cell, like Rho kinase a deletion, no vascular phenotype was reported for surviving Rho kinase h null animal. It is quite possible that Rho kinase a can compensate for h and vice versa. Alternatively, the major pathways governing Ca2+ sensitization in smooth muscle may not involve Rho kinase at all. We and others, have identified several other MYPT1Ks that phosphorylate MYPT1 at its inactivating site(s) in smooth muscles and other cells, including an endogenous MYPT1 associated kinase (MYPT1K/ZIP-like kinase/ZIPK/DAPK3 [4,35], DAPK1 DAPK2 ZIPK DRAK1 DRAK2

integrin-linked kinase [38] and myotonic dystrophy kinase [39,55,56]. Interestingly, like Rho kinase, all of these protein kinases phosphorylate CPI17 and LC20, in addition to MYPT1. The question that arises are: all of these protein kinases relevant in the regulation of SMPP-1M in all smooth muscles or restricted to certain muscle/cell types?

3. ZIP kinase ZIP kinase (ZIPK) was first cloned in 1998 as a cDNA recovered from a two-hybrid screen using mouse activating transcription factor (ATF, is a member of the cyclic AMPresponsive element-binding protein family of transcription factors) [57]. The human enzyme contains 454 amino acids and has a predicted molecular weight of 52.5 kDa (Fig. 2). It contains an N-terminal kinase domain of 263 amino acids spanning from residues 13 to 275 and an extended C-terminal domain of unknown function. The C-terminus

DAPK1 DAPK2 ZIPK DRAK1 DRAK2

----------------------------------------------MTVFRQENVDDYYD ------------------------------------MFQASMRSPNMEPFKQQKVEDFYD ----------------------------------------------MSTFRQEDVEDHYE MIPLEKPGSGGSSPGATSGSGRAGRGLSGPCRPPPPPQARGLLTEIRAVVRTEPFQDGYS ----------------MSRRRFDCRSISGLLTTTP-----------QIPIKMENFNNFYI .: : .:: * TGE--ELGSGQFAVVKKCREKSTGLQYAAKFIKKRRTKSSRRGVSREDIEREVSILKEIQ IGE--ELGSGQFAIVKKCREKSTGLEYAAKFIKKRQSRASRRGVSREEIEREVSILRQVL MGE--ELGSGQFAIVRKCRQKGTGKEYAAKFIKKRRLSSSRRGVSREEIEREVNILREIR LCPGRELGRGKFAVVRKCIKKDSGKEFAAKFMRKRRKGQD----CRMEIIHEIAVLELAQ LTS-KELGRGKFAVVRQCISKSTGQEYAAKFLKKRRRGQD----CRAEILHEIAVLELAK *** *:**:*::* .*.:* ::****::**: . .* :* :*: :*. -HPNVITLHEVYENKTDVILILELVAGGELFDFLAEK--ESLTEEEATEFLKQILNGVYY -HHNVITLHDVYENRTDVVLILELVSGGELFDFLAQK--ESLSEEEATSFIKQILDGVNY -HPNIITLHDIFENKTDVVLILELVSGGELFDFLAEK--ESLTEDEATQFLKQILDGVHY DNPWVINLHEVYETASEMILVLEYAAGGEIFDQCVADREEAFKEKDVQRLMRQILEGVHF SCPRVINLHEVYENTSEIILILEYAAGGEIFSLCLPELAEMVSENDVIRLIKQILEGVYY :*.**:::*. ::::*:** .:***:*. . * ..*.:. :::***:** : LHSLQIAHFDLKPENIMLLDRNVPKPRIKIIDFGLAHKIDFGNEFKNIFGTPEFVAPEIV LHTKKIAHFDLKPENIMLLDKNIPIPHIKLIDFGLAHEIEDGVEFKNIFGTPEFVAPEIV LHSKRIAHFDLKPENIMLLDKNVPNPRIKLIDFGIAHKIEAGNEFKNIFGTPEFVAPEIV LHTRDVVHLDLKPQNILLTSE-SPLGDIKIVDFGLSRILKNSEELREIMGTPEYVAPEIL LHQNNIVHLDLKPQNILLSSI-YPLGDIKIVDFGMSRKIGHACELREIMGTPEYLAPEIL ** :.*:****:**:* . * **::***::: : . *:::*:****::****: NYEPLGLEADMWSIGVITYILLSGASPFLGDTKQETLANVSAVNYEFEDEYFSNTSALAK NYEPLGLEADMWSIGVITYILLSGASPFLGDTKQETLANITAVSYDFDEEFFSQTSELAK NYEPLGLEADMWSIGVITYILLSGASPFLGETKQETLTNISAVNYDFDEEYFSNTSELAK SYDPISMATDMWSIGVLTYVMLTGISPFLGNDKQETFLNISQMNLSYSEEEFDVLSESAV NYDPITTATDMWNIGIIAYMLLTHTSPFVGEDNQETYLNISQVNVDYSEETFSSVSQLAT .*:*: :***.**:::*::*: ***:*: :*** *:: :. .:.:* *. * * DFIRRLLVKDPKKRMTIQDSLQHPWIKPKDTQQALSRKASAVNMEKFKKFAARKKWKQSV DFIRKLLVKETRKRLTIQEALRHPWITPVDNQQAMVRRESVVNLENFRKQYVRRRWKLSF DFIRRLLVKDPKRRMTIAQSLEHSWIKAIR-RRNVRGEDSGRKPERRRLKTTRLKEYTIK DFIRTLLVKKPEDRATAEECLKHPWLTQSSIQEPSFRMEKALEEANALQEGHSVPEINSD DFIQSLLVKNPEKRPTAEICLSHSWLQQWDFEN-LFHPEETSSSS--QTQDHSVRSSEDK ***: ****... * * .* *.*: .. . . RLISLCQRLS----RSFLSRSNMSVARSDDTLDEEDSFVMKAIIHAINDDNVPGLQHLLG SIVSLCNHLT----RSLMKKVHLRPDEDLRNCESDTEEDIARRKALHPRRRSSTS SHSSLPPNNS----YADFERFSKVLEEAAAAEEGLRELQRSRRLCHEDVEALAAIYEEKE TDKSETEESIVTEELIVVTSYTLGQCRQSEKEKMEQKAISKRFKFEEPLLQEIPGEFIY TSKSSCNGTCGDREDKENIPEDSSMVSKRFRFDDSLPNPHELVSDLLC * SLSNYDVNQPNKHGTPPLLIAAGCGNIQILQLLIKRGSRIDVQDKGGSNAVYWAARHGHV -----------------------------------------------------------AWYREESDSLGQDLRRLRQELLKTEALKRQAQEEAKGALLGTSGLKRRFSRLENRYEALA ---------------------------------------------------------------------------------------------------------------------

DAPK1 DAPK2 ZIPK DRAK1 DRAK2

DTLKFLSENKCPLDVKDKSGEMALHVAARYGHADVAQVQVLCSFGSNPNIQDKEEETPL 484 ----------------------------------------------------------KQVASEMRFVQDLVRALEQEKLQGVECGLR 454 -----------------------------------------------------------------------------------------------------------------------

DAPK1 DAPK2 ZIPK DRAK1 DRAK2 DAPK1 DAPK2 ZIPK DRAK1 DRAK2 DAPK1 DAPK2 ZIPK DRAK1 DRAK2 DAPK1 DAPK2 ZIPK DRAK1 DRAK2 DAPK1 DAPK2 ZIPK DRAK1 DRAK2 DAPK1 DAPK2 ZIPK DRAK1 DRAK2

14 24 14 60 33 72 82 72 116 88 129 139 129 176 148 189 199 189 235 207 249 259 249 295 267 309 319 308 355 324 365 370 364 414 372 425 400

Fig. 2. Sequence alignments of ZIPK with other members of the DAPK family. Alignments were made using sequences derived from Genbank using the ClustalW multiple sequence alignment program (http://www.ebi.ac.uk). (*) Indicates conserved residues in all five enzymes; (:) indicates conserved in at least three enzymes; (.) indicates semi-conserved residues in two or more enzymes. Sequence for DAPK1 is truncated at Leu 454.

T.A.J. Haystead / Cellular Signalling 17 (2005) 1313 – 1322

of ZIPK from 413 to 450 is predicted to form an alphahelical structure and shows homology to myosin heavy chain. In addition, a putative leucine zipper motif is present in the C-terminus from residues 427 to 441. Human ZIPK shows high homology in its catalytic domain to members of the death associated family of kinases (Fig. 2). There are five members of the death associated protein kinase family: DAPK1, DAPK2, ZIPK (DAPK3), DRAK1 and DRAK2. ZIPK shows 83% and 79% identity to DAPK1 and DAPK2, respectively, and shows ¨ 40% homology to DRAK1 and DRAK2 [57]. There is no significant homology in family members outside of the catalytic domain. In contrast to the DAPKs, ZIPK does not contain a death or calmodulinbinding domain suggesting its activity is regulated independently of Ca2+ [58,59]. ZIPK, like other members of the DAPK family, causes cell death upon over-expression in a variety of cell types suggesting the enzyme may play a role in apoptosis [57,60,61]. Cells that over-express ZIPK typically show signs of rounding, membrane blebbing, DNA fragmentation and detachment from the matrix. However, it remains unclear what the mechanism of cell death is as several indicators of apoptosis, such as caspase-3 activation or PARP cleavage have not been found with ZIPK over-expression [62]. It has been argued that cell death induced by ZIPK and other DAPK family members may be a result of autophagy as opposed to apoptosis [62]. Some insight into the phenotype caused by the overexpression of ZIPK may be gained from studying its candidate substrate proteins. ZIPK, like other members of the DAPK family, is capable of phosphorylating non-muscle myosin light chains [4,6,35,63,64]. Indeed, phosphorylation of myosin light chain is known to cause reorganization of the actin cytoskeleton and this could explain, at least in part, some of the cellular phenotypes observed with ZIPK overexpression such as membrane blebbing and cell rounding [7,65]. Initial indications in support of an involvement of ZIPK in the regulation of SMPP-1M came from studies by our laboratory searching for alternate MYPT1 kinases in smooth muscle [4]. A myofibrillar fraction was prepared from whole pig bladder smooth muscle and fractionated over an ATP affinity media we had designed for isolating protein kinases from complex mixtures, followed by anion-exchange chromatography [4]. Chromatographic fractions were assayed for SMPP-1M activity against 32P-labeled myosin and with recombinant MYPT1 for protein kinase activity that would phosphorylate the protein at T695. Two peaks of co-migrating phosphatase and kinase activity were observed. In-gel protein kinase assays were performed on active fractions and identified a distinct band of comigrating kinase activity at 32 kDa and a minor band at 54 kDa. Direct peptide sequencing identified the 32 kDa band as homologous to leucine zipper interacting protein kinase (ZIPK). At that time, we termed the enzyme ZIP-like kinase, because we had used a novel proteomic method called mixed peptide sequencing to identify the protein. The

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ZIPK sequence was only recently reported in the RIKEN database as a hypothetical cDNA clone (circa 1998). Subsequently, we have shown that ZIP-like kinase is indeed ZIPK; the 32 kDa protein originally identified is a proteolytic fragment of the full length 54 kDa form. ZIPK preferentially phosphorylates MYTP1 at its inactivating site T654 (chicken sequence), as well other sites including, T675 and S808 (chicken sequence) [4]. Addition of a recombinant constitutively active form of human ZIPK to permeabilized smooth muscles causes profound Ca2+ sensitization through direct phosphorylation of endogenous MYPT1 and inhibition of SMPP-1M activity [35]. In vitro, the K m for MYPT1 is ¨ 2 AM, about 15-fold lower than that determined for Rho kinase [4]. More recently, Mendelsohn and colleagues used high stringency screening of a human aortic cDNA library to identify the SMPP-1M associated kinase and identified 18 positive clones, all of which proved to be clones of hZIPK [66]. The authors also showed that full-length and N-terminal hZIPK bound the C-terminal domain (amino acids 681– 847) of MYPT1 and that hZIPK immunoprecipitated with the targeting subunit. Interestingly, the authors also showed that a dominant negative form of RhoA inhibited the hZIPK-MYPT1 interaction [66]. These data elude to a possible mechanism for RhoA in promoting the hZIPK-MYPT1 interaction. Although temporal and spatial location of activated RhoA at the plasma membrane relative to ZIPK (which is bound to MYPT1 on myosin) raise concerns over such a mechanism of regulation.

4. The regulation of ZIP kinase In our original paper reporting identification of ZIPK, we also showed that treatment of isolated smooth muscles with the Ca2+ sensitizing agonist carbachol caused activation and increased phosphorylation of the protein kinase [4]. Importantly, this activation was exacerbated by calyculin A (a potent inhibitor of PP1/PP2A class of phosphatases) and could be reversed by treatment of the immunoprecipitated kinase with purified protein phosphatases. Taken together, these findings strongly suggest that ZIPK activity is regulated by phosphorylation in smooth muscles. To comprehensively identify the important regulatory phosphorylation sites on ZIPK, we employed a combination of in vivo isotope labeling, mass spectrometry and site directed mutagenesis [67]. Seven phosphorylation sites in ZIPK were identified that regulate both its enzymatic activity and localization including T180, T225, T265, T299, T300, T306 and S311. Mutational analysis showed that phosphorylation of T180 in the kinase activation T loop, T225 in the substrate-binding groove and T265 in kinase subdomain X are essential for full ZIPK autophosphorylation and activity towards exogenous substrates. Abrogation of phosphorylation at T299, T306 and S311 had little effect on enzymatic activity but T299A and T300A mutations resulted in the redistributing of ZIPK from the cytosol to the nucleus. In

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contrast, a T299D mutation (to mimic phosphorylation) redistributed the enzyme to the cytoplasm. With the exception of T299/T300, all of the identified phosphorylation sites in ZIPK are conserved in the closely related DAPK2 and DAPK1. To understand the role of T180, T225, T265 in the regulation of ZIPK catalytic activity, a homology model was developed from the high resolution X-ray structure of the highly homologous protein DAPK (Fig. 3 and see Ref. [68]). The finding that ZIPK is phosphorylated at T180 shows that the enzyme bears all the hallmarks of a protein kinase whose activity is controlled by phosphorylation. T180 resides in subdomain VIII of the kinase activation ‘‘T loop’’, between the signature DFG and APE motifs found in all protein kinases (e.g. T180 is equivalent to T183 in MAP kinase or T286 in CamKII) (Fig. 3A) [69,70]. Upon phosphorylation, the T loop is thought to undergo a conformational change that stabilizes the kinase in an activated state [71 –73]. The importance of T180 in the regulation of ZIPK is supported by enzymatic studies in which the kinase domain of ZIPK only (ZIPKD273) autoactivates in the presence of ATP, and more convincingly by the finding that ZIPK T180A is completely resistant to activation. It remains to be determined if kinases other than ZIPK itself can phosphorylate ZIPK on T180 under conditions in which ZIPK may become activated (e.g. in the regulation of smooth muscle contraction). The roles of T225 and T265 in the regulation of ZIPK are less obvious; however, clues from the DAPK1 structure may include to possible function (Fig. 3B). T225 is expected to be part of an a helical region spanned by residues 221 – 233. Use of molecular accessible surfaces (Fig. 3B) showed that the gamma oxygen of threonine 225 has solvent accessibility, but is interestingly at the base of a modest pocket formed by a series of flanking residues (180 –183, 215– 230). T225 is removed from ATP such that direct interactions with ATP would not be expected. However, in inspecting published protein kinase X-ray structures, it was observed that the PKA catalytic subunit [74] has high structure homology to ZIPK/DAPK in the region of T225

A.

˚ 2 (ZIPK residues 180– (Fig. 3C) with a C-a rmsd of 0.36 A 183, 215 –230, PKA residues 201– 204, 236– 251). Moreover, this region can be seen to be directly involved in PKI peptide binding for PKA (Fig. 3C). These observations lead to the hypothesis that the activation of ZIPK due to T225 phosphorylation could be the result of several distinct effects on protein conformation, which in turn affect substrate binding. One plausible scenario would be related to a conformational rearrangement to facilitate substrate binding analogous to that seen for PKA. In particular, F239 of PKA has strong contacts with the bound peptide inhibitor. The homolog residue for ZIPK is L218 (Fig. 3C), but it has a different side chain rotamer conformation than F239. Phosphorylation of ZIPK T225 would place the phosphate group in close proximity and possibly in direct steric interference with L238. The adjacency of a hydrophobic leucine side chain with the charged phosphate group would be expected to be highly unfavorable. Fig. 3C shows a model in which the ZIPK backbone conformation in the region of residues 217– 223 is modified to a PKA like conformation. The conformation was readily attained, facilitated by the presence of G219, and places L218 into a conformation similar to PKA F239. Moreover, it provides good accessibility to the phosphate group, either to solvent or for a substrate side chain interaction. In summary, the effect of phosphorylation of T225 can be rationalized as potentially modifying the substrate-binding region into a favorable conformation to allow stronger contacts either directly with the phosphate group or adjacent residues. Phosphorylation of T265 also represents a novel regulatory site that may be unique to ZIPK and DAPK. Interestingly, phosphopeptide mapping studies of the T265A mutant showed that this site resulted in a loss of T180, T225, 306 and 311 sites, but not T299/T300 (67). These findings suggest a possible role for T265 as priming site for phosphorylation of other more distal sites. T265 is also conserved in DAPK1 and homology modeling studies show the amino acid is solvent accessible and resides in a conserved hydrophobic pocket that makes up part of the a helix of kinase subdomain XI (Fig. 3B). T265 is also

C.

B. T180 M201

T225

T265

Fig. 3. Homology models for ZIPK constructed from DAPK X-ray structure. The coordinates for DAPK1 were downloaded from the PDB database (1P4F). Figures were constructed using the Cn3D 4.1 software (http://www.ncbi.nlm.nih.gov/Structure/CN3D/cn3d.shtml). In A and B, yellow indicates each phosphorylation site. ATP in the active site of DAPK is shown as a ball and a stick model. The protein backbone is shown as tubes and side chains depicted in stick model format. C shows an overlay of ZIPK homology model with PKA-PKI complex [74]. Shown in green is the ZIPK peptide backbone for residues 180 – 183 and 215 – 230 with THR225 shown in cyan. The analogous PKA residues (201 – 204, 236 – 251) are shown in yellow. The PKI inhibitor is shown in ˚ of the PKI inhibitor. purple. The white surface represents the molecular accessible surface of PKA that is within 4 A

T.A.J. Haystead / Cellular Signalling 17 (2005) 1313 – 1322

positional placed close to the side chain oxygens of M201, which is itself part of the a helix of the T loop of subdomain VII. From the position of T265 within the DAPK1 structure, it is therefore possible that phosphorylation of T265 in ZIPK may exert a conformational change within the ZIPK structure to alter the position of the T loop to promote autophosphorylation at T180 and autoactivation. Mutagenesis studies showed that T299, T306 and S311 are not directly involved in the regulation ZIPK enzymatic activity. Individual mutation of T299, T306 or S311 to alanine resulted in only a modest increase in activity (¨ 1.5fold) when assayed for the ability to phosphorylate myosin light chain in vitro. Phosphopeptide mapping studies of each mutation showed a selective absence of a radioactive peak associated with each of the sites, without effect on other autophosphorylation sites. This finding contrasts mutations at T180, T225 and T265, which generally suppressed autophosphorylation at all sites. The finding that phosphorylation of ZIPK on S306 and S311 had no significant affect on enzyme activity is in agreement with previous findings for DAPK and DRP-1 (62). Residue S308 in DAPK or DRP-1 is the equivalent of S306 in ZIPK (Fig. 2). Although in two separate reports it was argued that phosphorylation of S308 in DAPK [75] and S308 in DRP-1 [62] was inhibitory, the phosphorylation site mutant of DAPK(S308A) or DRP1(S308A) did not show any difference in activity compared to wild-type enzyme in their ability to induce phosphorylation of myosin light chain in cells [62,75]. Also in agreement with our results is the finding that phosphorylation of DAPK at S313 (the equivalent of S311 in ZIPK) did not alter the catalytic activity of the enzyme towards myosin light chain [62]. Phosphorylation of T299 also does not have an appreciable affect on enzyme activity; however, it is involved in regulating the intracellular localization of ZIPK. Mutation of T299 and T300 to alanine results in localization of ZIPK to the nucleus compared to wild-type ZIPK, which is cytoplasmic (67). Consistent with this model, mutation of T299 to aspartic acid (to mimic phosphorylation) resulted in a cytoplasmic localization for ZIPK. These findings are in general agreement with Shani et al. [76] showing that phosphorylation of ZIPK results in its exclusion from the nucleus. Recently, Shani et al. described the phosphorylation of ZIPK by DAPK and the identification of several ZIPK phosphorylation sites [76]. In this report, it was shown that DAPK can phosphorylate ZIPK in vitro on T299 and potentially on five other sites including S309, S311, S312, S318 and S326. To address the function of these phosphorylation sites in vivo, all six sites were either mutated to alanine (6A) or aspartic acid (6D) to mimic phosphorylation. Substitution of alanine for all six of the phosphorylation sites blocked the ability of DAPK to promote apoptosis when co-expressed in HEK293 cells, implying that the kinase mediates its cellular effect through activation of ZIPK. The authors also show that the alanine and aspartic acid mutations at all six sites in ZIPK reduced and promoted

1319

its apoptotic effects respectively suggesting that phosphorylation of ZIPK at T299, S309, S311, S312, S318 and S326 is activating [76]. These results are in contrast with findings from our laboratory showing that substitution of alanine at T299, T306 or S311 had little effect on ZIPK activity in vitro or in vivo. However, in the report by Shani et al., the enzyme activity of the 6A or 6D mutant of ZIPK was not measured [76]. In addition, although we identified ZIPK autophosphorylation sites and not DAPK phosphorylation sites on ZIPK, we found no evidence for ZIPK phosphorylation in vivo at S309, S312, S318 or S326 by 32P-labeling and Edman degradation or mass spectrometry. One possible explanation for this discrepancy is that ZIPK does not undergo phosphorylation by DAPK in vivo under our conditions. Another explanation may be that, in the report by Shani et al. [76], none of the phosphorylation sites were directly identified but rather were inferred by mutagenesis. Although phosphorylation of the C terminal domain of ZIPK has no direct effect on ZIPK enzymatic activity, clearly the domain has the potential to regulate activity. Truncation of the C-terminal domain of ZIPK at 276 or 342 results in enzymatic activation as measured by the phosphorylation of myosin light chain. Furthermore, expression of the C terminal truncated enzyme (ZIPKD273 or ZIPKD342) in cultured cells greatly enhances its effects on cell death. Mutation of the C terminal leucine zipper motif also has dramatic effects on both the activity and localization of the enzyme. Previously, it was shown that mutation of the leucine zipper of ZIPK to alanines (V427A + V434A + L441A) resulted in loss of self-association, loss of autophosphorylation of the enzyme and resulted in a decreased ability to induce cell death when overexpressed in NIH3T3 cells [57]. In agreement with this report, we found that mutation of the leucine zipper motif severely compromised the ability of ZIPK to oligomerize in the context of the full-length enzyme. Surprisingly however, we also found that ZIPK did not require the leucine zipper or the entire C-terminal domain for that matter to selfassociate [67]. We also show that the leucine zipper mutant had decreased activity in vivo as measured by a cytoadherence assay. However, in contrast to the report by Kawai et al. [57], we found that mutation of the leucine zipper motif resulted in a 2 – 3-fold increase in the enzymatic activity of ZIPK towards myosin light chain. One explanation for this discrepancy may be that, in the previous report [57], no enzyme assays were performed to measure ZIPK activity. Our data suggests, therefore, that the oligomerization status of ZIPK may affect its enzymatic activity with the larger oligomeric forms being more active than ZIPK that cannot oligomerize. Interestingly, mutation of the leucine zipper showed decreased activity when expressed in cells as measured in cytoadherance assays [67]. One possible explanation for this observation is that the leucine zipper mutant was localized predominantly in the nucleus compared to a cytoplasmic localization for wild-type ZIPK. Thus, localization of ZIPK in the nucleus may prevent it

T.A.J. Haystead / Cellular Signalling 17 (2005) 1313 – 1322

from interacting with its physiological substrates in the cytoplasm (such as MYPT1 or myosin light chain) that may be involved in mediating loss of cytoadherence and cell death. In summary, ZIPK is primarily regulated by phosphorylation, which controls both its enzymatic activity and cellular localization. It should be noted that five of the phosphorylation sites identified in ZIPK are completely conserved in other members of the DAP kinase family, suggesting similar mechanisms of regulation are likely to be utilized by those enzymes (Fig. 2). For example, T180 is conserved in the DAPK family and, given that ZIPK shares 80% identity within its catalytic domain with DAPK1 and DRP, it seems highly probable that activation of these enzymes will also require phosphorylation of this residue. The C-terminal domain suppresses ZIPK enzymatic activity and loss of this domain results in constitutive activation and increased cell death when expressed in cells. The domain also contains a leucine zipper motif that enables oligomerization with itself and is involved in cytoplasmic localization within cells. The leucine zipper is also a site for interaction with MYPT1 or other regulatory proteins. Although the C domain is an attractive target for regulation, direct evidence for control of ZIPK activity through this domain other than through proteolysis has yet to be demonstrated. Given the extensive evidence for regulation of ZIPK, in smooth muscles, how might Ca2+ sensitizing agonists activate or regulate enzyme? Fig. 4 outlines two possible scenarios for the regulation of ZIPK. First, ZIPK is regulated by a ZIPK kinase kinase that phosphorylates and activates the enzyme at T180, T225 and T265. Data from our laboratory suggests all three sites would be required to activate the enzyme. A likely upstream activating kinase is Rho kinase; however, ZIPK is a poor substrate for Rho kinase in vitro [4]. Shani et al. have reported that over-expression of DAPK1 in cells results in activation of ZIPK, suggesting that DAPK is a ZIP kinase kinase [76]. However, in our hands, the major DAPK phosphorylation

site on ZIPK T306 is not activating. Furthermore, at least in smooth muscle, increases in intracellular Ca2+ do not activate ZIPK. If ZIPK was a target for DAPK1 in vivo such a mechanism might be predicted, since DAPK1 is itself regulated by Ca2+ through its calmodulin-binding domain [8]. The second possibility is that factors acting directly on ZIPK promote its activation through autophosphorylation. As discussed, inactive recombinant mutants of RhoA have been demonstrated to prevent binding of ZIPK to MYPT1 [66]. In non-muscle cells, MYPT1K(ZIPK) appears to be localized in the nucleus until an apoptotic signal is initiated, which then causes the protein kinase to complex with other proteins and translocate to the actin filament system. A likely candidate site for regulating this event is phosphorylation at T299. PAR4 has also been identified as a possible binding partner that may be involved in the relocalization of ZIPK from the nucleus to the cytoplasm [77].

5. ZIPK as a therapeutic target

non-muscle cells

From the discussions above, ZIPK is clearly emerging as having highly selective actions in cells centered around Ca2+ independent regulation of myosin light chain phosphorylation in smooth muscle and non-muscle cells. Since ZIPK belongs to a relatively small and unique class of serine/ threonine protein kinases, the enzyme may therefore represent an ideal target for the development of a new generation of antihypertensive or antiasthmatic drugs. Small molecule inhibitors of protein kinases are thought have great potential for the development of new pharmaceuticals to treat a variety of human diseases from cancer, diabetes, inflammation and cardiovascular disease [78 –80]. Indeed, at the time of writing, at least one protein kinase inhibitor is being used as a therapeutic (Gleevac) for the treatment for chronic myeloid leukemia and many others inhibitors are in late stage clinical development for the treatment of other cancers [81 –84]. Major concerns over inhibitors of ZIPK

nucleus PAR4

? Pi

ATP

T299/300

T299/300 kinase

S113

kinase domain ? unknown factor promoting autophosphorylation

T180

LZ T225

T265

T306

RhoA

?

ZIPkinase kinase

? ? Rhokinase

MYPT1

smooth muscle

1320

RhoA Fig. 4. Current model depicting possible mechanisms of regulation of ZIPK in non-muscle and smooth muscle cells.

T.A.J. Haystead / Cellular Signalling 17 (2005) 1313 – 1322

are that they might target the closely related DAPKs or if ZIPK is involved in the regulation of apoptosis, and cause uncontrolled cellular proliferation. Although genetic studies are required to address the later issue, inspection of the sequences of DAPK1 and ZIPK within the ATP-binding site suggest there is room for the development of selective inhibitors, despite their close identity. Inspection of the DAPK structure with ATP analog bound shows that there are 15 amino acids that make direct contact with the nucleotide (70). Although these residues are completely conserved in ZIPK, there is sequence variance in two regions of the ATP-binding pocket that suggests that the internal molecular space of the ATP-binding pocket of ZIPK may be somewhat different than in DAPK1. The first region of variance occurs between sub domain I containing the conserved GXXGXG motif and sub domain II containing the conserved Lys42 residue. The solution structure of DAPK1 shows several residues within this domain are solvent accessible in the ATP-binding pocket and not are conserved in ZIPK, e.g. R28, Q31, Q32, G35, M37 and E38. The second region of variance is in subdomain VI namely R215, E217, A220 and S222 (Fig. 2). In addition to these regions of variance, there are other non-conserved solvent accessible amino acids that are also likely to introduce distinct differences in the internal molecular space within the ATP-binding pocket of ZIPK compared with DAPK1 despite their overall sequence identity, including D125, H128, K133, H150 and A152. At least one group has developed selective inhibitors for DAPK1 based on a 3amino-6-phenyl pyridazine scaffold (Velentza et al. [85,86]). These inhibitors may be a promising starting point for the development of ZIPK inhibitors since they show a reasonable degree of selectivity for DAPK1 relative to the next closest family Ca2+/calmodulin dependent protein kinases such as MLCK and CAMKs.

Acknowledgements Thanks to Elizabeth Synder for the preparation of figures and James Veal (Serenex Inc., Durham, NC) for help with ZIPK phosphorylation site modeling studies.

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