TIBS 12- February 1987 53 Open Question: Proteolytic activation of protein kinase C: a physiological reaction? Andrew W. Murray,AgnesFoumier and StephenJ. Hardy tVotem ianase C aanaty esrevoszbly reg, d,m,d by ckacylglyceml transtonty generated by spe- crfic receptor buulmg or by phorbol esters Proteolyttc acnvanon of the lanase also occurs m intact cells incubated web phorbol esters, raising the posstbday of new funazons for des key enz~e The involvement of a Ca 2+ and phos- phohptd-dependent protein lunase (pro- tern lunase C, PK-C) m transmembrane signalling ts now well estabhshed I-3 The kmase ts actwated by 1,2-dtacyl- glycerol (DAG) tranmently generated as a consequence of the receptor-mediated hydrolysis of mosttol phosphohplds by phosphodlesterase attack The other products of hydrolysas, the mositol phos- phates, are probably revolved m the mobthzatton of Ca 2+ from mtracellular stores In a number of systems at least, Ca2+ mobthzatlon and PK-C acuvanon appear to act synergmt~cally m generat- mg a particular btolog~cal responsela. 4 Detmls of the mechamsm for the acuva- uon of PK-C ate not yet clearly under- stood It is generally accepted that DAG (or active phorbol esters wluch act as DAG analogues) stab]hze a quaternary complex between the enzyme, Ca 2+ and membrane phospholiptd. In some sys- tems, elevation ofcytosohc Ca 2+ concen- trations may enable PK-C to be acavated at lower concentrations of the s~gnal molecule (DAG or phorbol ester) 5-7 Protein lunase C can also be irrever- sibly actwated by proteolysm In fact the enzyme from rat brmn was ongmally reported as a soluble proenzyme which was activated by mcubatmn with a Ca 2+ protemease, also purified from brain s The soluble proenzyme (PK-C) was sub- sequently shown to be acavated by Ca 2+ and phosphohpld and, at low Ca 2+ con- centrations, to be stimulated by DAG The proteolyacally acavated form of PK-C (called PK-M by the ongmal authors) had a molecular mass of about 50 kDa and was catalyacally actwe m the absence of Ca 2+ and phosphohpld 9 For- A W Murray, A FourmerandS J Hardyareatthe School of BtolohccalSc,x, nces, FImdersUmvenny. BedfordPark, South Australia,5042 matlon of PK-M was strongly enhanced by DAG and phosphohptd, indicating that the active, membrane-associated form of PK-C was the preferred sub- strate for proteolysts9 Although the proteolyttc actwatton of several protein kinase enzymes is well known (for references, see Refs 10 and 11) tt Is not fashionable to as~,~.be physiological function to tins process However, over the last few years evi- dence has been obtmned that proteolyt~c acavatton of PK-C also occurs in several intact cell types incubated with the tumour-promotmg phorbol ester TPA, the two best characterized systems being human platelets and neutrophds Incu- bation of platelets with TPA resulted m the formauon of a 50 kDa protein lunase, active m the absence of added Ca2+ and phosphohpld, which eluted from DE52 cellulose at a higher salt con- centratton than PK-C 12.13 Formation of this lunase was blocked by pre-mcubat- mg the platelets with leupeptm, a known mhthttor of the CaZ+-dependent neutral protemase which cleaves PK-C In addi- tion, phosphorylatton of myoan regula- tory hght chmns by both PK-C and the 50 kDa kmase, followed by analysts of the tryptlc pepttdes, indicated a smu- lar pattern of phosphorylatton by both kanases I3 Sinularly, incabation of human neutrophlls with TPA for 10 nun resulted m an almost quantttatwe con- version of PK-C to an enzyme of ~50 kDa wluch was catalytically active m the absence of Ca2+ and phosphohpldl4 The independent form of the enzyme accumulated predonunantly in the cytosohc fracuon, and its formauon could be blocked by leupeptm in earlier m vztro studies the same group had shown that m the presence of Ca 2+, both PK-C and a Caa+-dependent proteinase isolated from neutropluls associated with neutrophd membranes ts When the two enzymes were assoaated with the mem- brane, PK-C was cleaved to a PK-C form independent of Ca 2+ and phospholipld. that was then released from the mem- brane (Fig 1) Formatmn of lower M r forms of PK-C have also been reported m human B lymphocytes incubated with TPA together with the Ca 2+ tonophore tonomycm 16, and m human breast cancer cells incubated with TPA 17 The most obwous explanation for these results is that TPA stabd~zes the membrane assocmuon (and acuvauon) of PK-C wluch m then subJect to proteolysts by a Cae+-reqmnng neutral protlnease, followed by release of the catalytic frag- ment into the cytosol Tlus process pre- sumably accounts for the widespread observation that prolonged incubation of many cell types with phorbol esters results m the depleuon of total cellular protein lunase C acawty and m resist- ance to many phorbol ester responses is-21 in these longer term experiments no evi- dence of a lower M r form of PK-C has been observed, and ~t may be that the catalytic fragment is itself subsequently inactivated Although TPA provides a caricature of the normal situation by causing mas- swe association of PK-C with mem- branes, it would be predicted that natu- ral hgands which stimulate inosttol phos- phohptd turnover and activation of PK- C would also cause the formation of small amounts of the catalytic fragment The complete nutogens bombesm and PDGF are of particular interest, as both growth factors have been shown to acti- vate PK-C m target cells 22--'4 The key question, therefore. Is whether this reac- uon has physiological significance or whether proteolysts simply provides a mechanism for the irreversible tnattl~a- tton of the active form of this enzyme Although only limited data Is available, the answer appears to be complex, with some responses clearly requiring native enzyme and a hint that others may involve its proteolysts product For example, the ability of TPA to induce oxygen radicals and the release of a scnne protemase from human neu- trophds was blocked by mhtbltors of PK- C and enhanced by leupeptm ~ hmch pre- vented its proteolyms~-~ It was therefore concluded that these two responses were mediated by membrane-associated native PK-C On the other hand El~ewer Science Pubh~her~ B V Am~erdam i}~tTh - ~ObT/g7,t$1)2011