Growth
factors and cell proliferation Enrique
Imperial
Cancer
Rozengurt
Research
Fund, London,
UK
New advances in the identification of ligands and signal transduction events that induce cell proliferation through tyrosine kinase and C-protein-coupled receptors have extended our understanding of the pathways that control cell proliferation by growth factors. Current
Opinion
in Cell Biology,
Introduction
[I*] ). III every case. the formation of stable complexes requires the yrosine kinase activity of the receptor. The association of qtoplasmic proteins with the activated receptors is mediated by a non-catalytic domain of w 100 amino acids, the src homology (SH)Z region (revicared in [2-e] 1. These domains, expressed ;I$ bacterial fusion proteins, are sufficient to fomi stable complexes in lttt-0 with the liganded receptors [3*,9]. It has been proposed that vrosine l~tiospho~~lation of specific residues in the poljpeptide chain of the receptor acts as a switch to induce high-aJ%niv binding of SHZ-containing cytoplasmic proteins. Differences in the amino acid sequences between SH2 domains appear to result in distinct binding affinities [ 2**] Hence, SH2 domains plrly a crucial role in the association of q~oplasmic proteins with cellular and receptor h’rosine kinases.
The proliferation of most cells can he stimulated by ;t variety of extracellular ligands that act in a combinatorial and q~lcrgistic f&lion. These factors bind to receptors located on the cell surfrice and acti\.ate multiple signalling path\~iys. This re\,ie\v \vill focus on the earl!. c\‘ents in the action of gro\\Th factors. Lritcr steps in the cell cycle, in. eluding the role of 133-i~(‘~~aiid cyclins, will he the subject of 3 sepzirate section. At lczst two major signal-transduction l-xtth”‘a)~s initiate ca~~cades of molecular events leading to cell proliferation: one involves pol!peptide growth t:,ictors [e.g. pluteletderived growth ktc’tor ( PDGF) and epidermal growth Etctor ( EGF) ] that bind to receptors with intrinsic vrosine kinnse :lcti\it) :ind the other invohes receptors coupled by 8l[:liiine-nl[cIeoticle binding proteins (G proteins 1 to etfector activation. During the past year considerable Xl. vaiices have been made in the identifcation of extracelMar frictors :ind in the elucidation of signal-transduction c\‘ents that induc~e cell proliferation through both the t\‘rosinc kinase :md the G l”otein-mccliated path\~i!.s.
Tyrosine Signal
kinase
1992, 4:161-165
It is known that PDGF and EGF receptors associate with and phosphoqlatc PLC-y,, but the precise role of this moclifcrltion remains unclear. Recent evidence demonstr;ltes that PLC-), phosphorylation is directly responsible for the activation of this enqme in li\ring cells. Thus, o\~erexpression of PLC-y, and PLC-y1 increases tyrosine phosl~ho~lation of these enzymes and inositol phosphatc formation in response to PDGF [ 51. The mechanism of this acti~ltion was investigated by substituting phen!U:lnine for qnjsine at phosphoyl:ltion sites 77 1, ‘83 and 125-r, aiid expressing the mutant enqmes in 3T3 cells. Phen~~l:tlanine substitution ;Lt Tyr7H3 completely blocked the accumulation of inositol phosphates induced b!r PDGF 16**]. These results provide direct e\idence that the PDGF receptor stimulates the function of ;m intracellular signal-transducing protein by site-specilic n’rosine I~hosphon’lation.
receptors
transduction
There is consiclerablc e\~iclence th:it poI)~qXicle gro\\Th fnctors. including PDGF and EGF. activate their corresponding receptors 1~). inducing receptor dimerization and subsequent transl~hosl~ho~,t~itioi~ at specific vrosine residues. These liganclccl receptors ph!.sic:lll!. :wsociatc with mcl phosplloIyl~ltc :i set of q~op’lasmic proteins iniplic:itecl in intracellular sign:il transduction P;lth~~l)s. These include the y, isoform of I”)l}l’hosphoinositidespecifc l~hosl~holil~;is~ C ( PLC-y, ). l~‘hosl~1iatid!~lii~ositol 3’ kinase (Pl3Ii ). 1~21 U’S GTP;ise-;1cti\.;lting protein (GAP ). t)rrosinc kinases of the Src fiiniil)~ and other cellular proteins that have not. ;is !rct, been identified ( re\iciwecl in
The association
of Pl3K with PDGF receptors has continattention. This enzyme phosphotylates the inositol ring of I’hosptlLltidylinositol at the D3 position and is con~l~osed of t\vo subunits with apparent m&cular m:lsses of 85 and 1 10 kD [ 71. The 85 kD subunit has been cloned in Merent laboratories and shown to LIC’CI to ;lttl’act
Abbreviations BDNF-brain-derived
HCFphepatocyte PDCF-
growth
platelet-derived
neurotrophic factor; factor; NFl--neurofibromatosls growth
EGF
efxlermal growth factor; gene product; NCF-nerve
factor; Pl3K-phosphatldyllnosltol SCLC ~-small cell lung cancer;
@
Current
Biology
SH-src
Ltd
(;AP~~~CTPase-activating growth
3’kinase; PKC-protein homology domain.
ISSN
0955-0674
factor; kinase
protein; NT-3-neurotrophin-3;
C;
PL-phospholipase;
161
162
Cell multiplication
. contain SH2 domains that mediate the association of PI3K to phosphorylated receptors, but lacks catalytic activity [8*-lo=]. A synthetic peptide of 20 amino acids representing a conserved region of the kinase insert domain blocked the interaction in zpifro of the PI3K with the phosphorylated j%PDGF receptor [ 111. The precise biological significance of PI3K remains unknown. It has been thought, however, that it plays a role in eliciting cell proliferation, as deletion mutants in the p-PDGF receptors abolish both PI3K association and DNA .synthesis [lo]. In contrast, a deletion mutant of the a-PDGF receptor, lacking 80 amino acids of the kinase insert region, failed to show receptor-associated PI3K but retained partial mitogenic activity [ 121. Furthermore, mutations in the a-PDGF receptor that markedly impair its association with PUK did not prevent PDGF-induced mitogenesis [ 13**]. This demonstrates that the association of PI3K to a-PDGF receptor is not required for PDGF-induced mitogenic signalling. Future experiments should elucidate whether this constitutes a crucial difference in the mechanism of action of a- and P-PDGF receptors. The upstream and downstream mechanisms by which mitogenic stimuli engage ~21 rs in cellular signalling remain poorly understood. The findings that GAP associates with and serves as a substrate for phosphotylation by liganded tyrosine kinase receptors suggested a link between receptors and p2Ira mediated by GAP, but substantial evidence was lacking. It is known that phosphorylated GAP associates with two other cellular proteins, p62 and ~190. EGF treatment stimulates GAP phosphotylation and induces complex formation between GAP and ~190 [ 14.1. Interestingly, this complex has reduced GAP activity. This finding suggests that GAP phosphorylation plays a role in the regulation of p21raq activity. A region of the neurofibromatosis gene product (NFl) that has sequence similarity to the catalytic domain of GAP also stimulates p2 1 ra$ GTPase activity. Interestingly, NFI and GAP are regulated in l&-o by lipid mediators, particularly arachidonic and phosphatidic acid [ 15*,16]. Elucidation of the physiological significance of these effects in the regulation of p2I ra requires further experimental work. Ligands
for Trk, met and kit
During the past year, a number of ligand-receptor systems have been elucidated. The Trk family of tyrosine kinases has been identified as the cellular receptors for the neurotrophins, consisting of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3). The product of the proto-oncogene Trk serves as a high-affinity receptor for NGF [17-l whereas T&B, which exhibits a high degree of homology to Trk, has been identified as the receptor for BDNF [W-21*]. NT-3 can bind to both Trk and T&B, as well as to a novel member of the Trk receptor family, TrkC, that does not recognize either NGF or BDNF [ 22.1. These studies demonstrated that a previously identified NGFbinding protein, known as the low-affinity NGF receptor is not required for neurotrophin-mediated signal transduction. In common with other activated growth factor receptors, binding of NGF to Trk rapidly increases the phosphotylation of PLC-y in responsive cells [ 231. These
studies constitute an important step towards understanding the signal transduction pathways that mediate the biological effects of the neurotrophins. Another series of studies revealed a relationship between hepatocyte growth factor (HGF), scatter factor and the c-met proto-oncogene tyrosine kinase receptor. HGF is a potent mitogen for hepatocytes in culture and promotes liver regeneration after partial hepatectomy. HGF also stimulates the proliferation of endothelial and epithelial cells [ 241. Independently, an activity that stimulated the motility of certain epithelial cells (e.g. Madin-Darby canine kidney cell line) had been isolated and termed scatter factor (reviewed in [25*] 1. The sequences of cDNA clones encoding these polypeptides were virtually identical [26-l. Moreover, both purified HGF and scatter factor bind and stimulate tyrosine phosphotylation of c-nzef [26*,27*]. Thus, scatter factor and HGF are the same polypeptide and are the ligands for the c-met tyrosine kinase receptor that mediates the stimulation of cell motility or proliferation in the corresponding target cells. An important development has been the identification of the product of the Steel locus of the mouse as a growth factor. It was known that mutations in this loc~~saRect the development of a variety of cell lineages and that the Sfeel and the \V%ite Spotti,zg loci are distinct genes with closely related functions (reviewed in [ 281). The Steel growth factor has been purified from several sources and shown to stimulate the proliferation of mast [ 291 and hemopoietic cells [ 30). This novel growth factor has been identified as the ligand for the c-kit proto-oncogene receptor tyrosine kinase, which is encoded by the Wite Spolfiq locus. Addition of Steel growth factor to normal mast cells stimulates rapid tyrosine phosphorylation of c-kit and induces the association of PI3K and PLC-7, to the liganded receptor [31*].
G-protein-coupled Ligands
receptors
and receptors
An increasing number of neuropeptides and vasoactive peptides (e.g. bombesin, vasopressin, bradykinin, endothelin and thrombin) have been shown to act as potent cellular growth factors and have been implicated in a vdriety of normal and abnormal biological processes including development and tumorigenesis. During the past year, an important finding has been that the colony growth of small cell lung cancer (SCLC) cells is stimulated by multiple neuropeptides, including bombesin, bradykinin, cholecystokinin, neurotensin and vasopressin [32.*]. Further studies showed that galanin acts as a cellular growth factor for SCLC cells [ 33.1. These novel findings are important because they suggest that the previously identified autocrine growth loop of bombesin-like peptides is only one of an extensive network of paracrine and autocrine circuits that sustain the proliferation of SCLC. The cloning and sequencing of the cDNA for several mitogenic peptide receptors including bombesin [ 3d*], neuromedin-B [ 35.1, bradykinin [ 36.1, thrombin [37.01 and vasoactive intestinal peptide [38] has been accomplished during the past year. The fundamental struc-
Growth
tural feature of these G-protein-coupled receptors the seven putative transmembrane domains - has been found in every case. Nevertheless, the expression cloning of the thrombin receptor revealed a novel mechanism of receptor activation. The physiological &and of the receptor is generated by proteolytic cleavage of the aminoterminal extracellular domain of the receptor [37**]. It will be interesting to determine whether all biological effects of thrombin are initiated by the same receptor or by other receptor subtypes.
Signal transduction
Many neuropeptide receptors are known to rapidly stimulate the PLC-mediated hydrolysis of polyphosphoinositides, but the molecular identity of the phospholipases and G proteins involved remains elusive. Although there is direct evidence indicating that PLC-?/ is a target for growth factor receptors with intrinsic tyrosine kinase activity (see the preceding section), a different PLC isoform, namely PLC-p, has been shown to be regulated by G proteins. This PLC isoform does not contain SH2 domains. An important development in delineating this signal transduction pathway has been the purification of novel heterotrimeric G proteins of the Gq subfamily. When incorporated into phospholipid vesicles together with PLC-p, the guanine nucleotide regulation of this PLC isoform was reconstituted [3!+41]. A role for PLC-p and Gq in the mechanism of action of growth-promoting peptides is plausible, but definitive evidence is not yet available. In contrast, GTPase-inhibiting mutations of ai.2, which were found in certain endocrine turnours, have been shown to convert this pertussis toxin-sensitive G protein into a stimulator of cell proliferation (42.1. Elucidation of the precise mitogenic signal transduction pathway activated by this mutant G protein requires further experimental work. The activation of PLC-y and PLC-p (as well as that of phosphatidylcholine-specific PLC or PLD) by polypeptide growth factor and mitogenic peptides generates diacylglycerol, the second messenger that activates protein kinase C (PKC). Because the role of this kinase family in cell proliferation is well established, it is essential to identify and characterize its physiological substrates. An acidic, myristoylated protein that migrates with an apparent molecular mass of 80 kD has been identified as one of the most prominent substrates for PKC, both in cultured cells and in several tissues. Recently, the cDNAs encoding the substrates from rat brain [43*], Swiss 3T3 cells [44**], murine macrophage (451 and human brain [46] have been cloned. Comparison of the rodent sequences with those of the PKC substrates from human and bovine brain (termed MARCKS) revealed considerable sequence divergence, suggesting either a low level of evolutionary pressure or the existence of a gene family. Interestingly, PKC activation causes a dramatic down-regulation of the expression of mRNA and protein of the 8OkD substrate from Swiss 3T3 cells through a post-transcriptional mechanism [44**]. This novel finding suggests that this PKC substrate, which is a calmodulin and actin-binding protein, may play a suppressor role in the control of cell proliferation.
factors
and cell proliferation
Rozengurt
Activation of neuropeptide receptors linked to rapid polyphosphoinositide breakdown does not necessarily lead to identical long-term responses. Thus, bombesin induces a striking and sustained release of arachidonic acid and eicosanoid production in 3T3 cells, whereas vasopressin, bradykinin and endothelin fail to stimulate a similar response [47**]. Toxins have provided useful tools for dissecting signaltransduction pathways in eukaryotes. Mastoparan, a tetradecapeptide that directly stimulates G protein activity, induces arachidonic acid release and stimulates DNA synthesis in synergy with other factors in 3T3 cells [48*]. Pasteurellu multocida toxin, a 145kD protein, is a potent mitogen for cultured libroblasts. It induces striking polyphosphoinositide breakdown [49”] and constitutes the first intracellularly acting bacterial toxin that has been identified to activate this signal transduction pathway. Concluding
remarks
A striking feature of the diverse tyrosine kinase receptors summarized here is that in response to ligand binding (e.g. PDGF, EGF, libroblast growth factor, NGF, BDNF, NT-3 and, Steel growth factor), they rapidly associate with a specific set of SH2containing cytoplasmic proteins. An important challenge for the future is to determine whether the discrete number of cellular targets identified so far can account for all the molecular events (ionic fluxes, protein phosphotylation, gene expression etc) and biological responses induced by activation of these receptors. Classically, tyrosine phosphorylation by polypeptide growth factors and the generation of second messengers via neuropeptide receptors linked to G proteins have been regarded as separate though complementaty pathways in the mitogenic stimulation of cells. The recent demonstration that bombesin, vasopressin and endothelin stimulate tyrosine phosphorylation of specific substrates in Swiss 3T3 cells suggests the existence of an additional signal transduction pathway in the mitogenic response to these agents [50**]. Indeed, these peptides do not stimulate tyrosine phosphotylation of PLC-yt, PISK or GAP. Elucidation of the components of such a pathway, as well as the identity and function of the kinases and substrates, warrants further experimental work. References
and recommended
Papers of particular interest, published view, have been highlighted as: . of special interest .. of outstanding interest I. .
the annual
period
of re-
CNLEY LC, AUGER KR, CAHPENTEH C, Ducwotrnr B, GRAZ~ANI A, KAI’ELLER R, SOLTOFF S: Oncogenes and Signal Transduc-
tion. Detailed sociation
within
reading
Cell 1991, &281-302.
review on receptor tyrosine kinases and particularly on the asof wious q~oplasmic proteins with tyrosine kinase receptor.
Kocti CA, ANDEKON D, MORAN MF, Ettts C. PAWSON T: SH2 and SH3 Domains: Elements that Control Interactions of Cytoplasmic Signalling Roteins. Scicwce 1991, 252:66%674. in important review on the evidence for the role of SH2 domains in linking cellular proteins at receptor tyrosine kinases.
2. ..
163
164
Cell multiplication .
3. .
ANDERSON D, KOCH CA, G&7 I EUS C, Mot&v Binding of SH2 Domains of Phospholipase Src to Activated Growth Factor Receptors.
MF, PA~XON T: Cyl, GAP. and Scietzcc, 1990,
250:97%982. SH2 domains 4.
5.
can directly
bind
lig.mded
EGF
and PDGF
receptors.
MOHAMMADI M, HONEGGER AM, ROTIN D. FIXZHER R. BEILOT F. LJ W, DIONNE CA, JA~F M, RLIBINSTEIN M, SCIUSSINGER J: A Tyrosine-phosphorylated Carboxy-terminal Peptide of the Fibroblast Growth Factor Receptor (Fig) is a Binding Site for the SH2 Domain of Phospholipase C-yl. A/o/ Cc// Hiol 1991. 11:5068-5078. Su~liw.4~ I+ Euls C, LIN L-l PAWON T, KNOW J: Plateletderived Growth Factor Increases the In Vitro Activity of Phospholipase C-y, and Phospholipase C-y?. :I101 Cell Rio1 1991. 31:201~2025.
6. ..
K~hr HK, Klh! JW. ~ll~3~R5ll3N A, MAR R. 1151)&v J, Torn’ N. Shlllll AD, h1OR64\ SJ, COL~RTNEII~GE SA, PARKI~R PJ. WATE~I~X~) MJ: Characterization of Two 85 kd Proteins that Associate with Receptor Tyrosine Kinases, Middle-T/ppbOC-SrC Complexes. and PI 3. kinase. Cc// 1991. 65:91-IO+. This paper and [8*.9-l describe the purification and cloning through di&renr routes of the Hi kD subunit of P13K;. and drmonstnlte the presence of SH2 domains and binding of this protein IO liganded yrosine kinase receptors. 11.
12.
01-s~
THOhIP.sOI\I
ESCOHEDO JA, KAI~IIV\: DR, KA\;~~A~x;H WM. TI:RCK CW. WIUU!S LT: A Phosphatidylinositol-3 Kinase Binds fo Platelet-derived Growth Factor Receptors Through a Specific Receptor Sequence Containing Phosphovrosine. .1/o/ Cell Rio/ 1991, 11:1125-1132. HEIL>AKA~ MA. I’IERCE JH. lUhlBAKDl D, Rwc;~ew M. GUUXI JS, MATXII T, AARONSON SA: Deletion or Substitution Within the a Platelet-derived Growth Factor Receptor Kinase Insert Domain: Effects on Functional Coupling with Intracellular SignaIling Pathways. .1/o/ Cell Hiol 1991. 11:13+l-r2.
13. ..
Yll J-C, HEIIXRAN MA, PEIRCE JH, GL:XINI) JS, Lohle#.ni D. RLIGGI~RO M, AAKONSON SA: Tyrosine Mutations Within the a Platelet-derived Growth Factor Receptor Kinase Insert Domain Abrogate Receptor-associated Phosphatidylinositol3 Kinase Activity Without AITecting Mitogenic or Chemotactic Signal Transduction. ,llo/ Cell Biol 1331. 1 1:378C-3785. This paper suggese that Pl3K association wirh the a-PDGF receptor can be dissociated from mirogenesis. 13. .
MORAN IMF, POWS P, MCCO~L\IICK F, PAU’S~N T. EUJS C: Protein-tyrosine Kinases Regulate the Phosphorylation, Protein Interactions, Subcellular Distribution and Activity of ~21~ GTPase-activating Protein. ;Ilo/ Cc// Rio/ 1991, 11:1804-1812.
M: The Growth
65:89i-903. 19. .
10. .
E. BAIUACII) for Nerve
SOPPET D. EXXNIX)N E. MARN;OS J. MII~I)Il:h~~ DS, Rrw SW, BLAIR J, BLIRTON LE. STANTON BR. KAPIAN DR. IlwrxR T. NIKOIJCS K, I’Ak\l)r\ LF: The Neurotrophic Factors Brainderived Neurotrophic Factor and Neurotrophin-3 are Ligands for the lrkB Tyrosine Kinase Receptor. Cc>// 1991.
122-l.
9.
it.5 as
Goi.~wc M. TANAKA K. DOBROW~OLSKI S. WOOD D. Tw MH, MAR+LUL M. TA~MNOI F. STACEY DW: The GTPase Stimulatory Activities of the Neurolibromatosis Type 1 and the Yeast IRA2 Proteins are Inhibited by Arachidonic Acid. El/R0 ./ 1991. 10:298’-2903. Prwidrs elidewe that GAP and NFl are regulated itr lY/ro by lipid mcxiiators.
SW
.
is fo enhance
15. .
KR. COHEN B. and CharacterRat Liver. ./ Biol
7.
CAlU’ENlXR CL, DLIch~ORTH BC, AL’GER SCHAFFHAI!SE~ BS, CAXIUX LC: Purification ization of Phosphoinositide 3-kinase from C%rtn 1990, 32:1970-r-1971 1.
Emphasizes that the role ol’ GAF’ phosphorylation sociation with another cellular protein. ~190.
V. JINC S. L~w~hi~e F. T,~I.I~ P. BKYANI C, JONES KR. &ICHARI\KI)T LF. BruwcIi) M: Protein Kinase is a Receptor for BrainFactor and Neurotrophin-3. Cell 1991,
GWSS DJ. N~E SIH. I l.~~-LWPOltOs I’, MACC~II MJ. SQI’ISIO $I’. G7080. Reports the molecular cloning of the 80 kD PKC substrate from rat brain and reveals a novel post-translational modification.
Sc‘i 1 ‘SA 1901, 88:772+772H. Reports
Biof
43.
K: Expression
R\Y’. JAWAGIN
Receptor.
and Characterthe pl lsozyme C. Identifica-
d-3. .
rccepror.
35.
36.
A.
PETEKWN
of the Human /+DC N&l AC&
KE,
GOETXL
Vasoactive
.Sci LISA
EJ:
In1991,
Through a novel mt~hanism, Alsrelrre//~~ togen. is shown to induce the prtxluction identical to those induced by bombesin. 50. ..
multocidrc of inositol
toxin, a potent miphosphate species
%~L.WY I. GII. J. ~H%L+!!N W. SINNE’ITSMITH J. ROZENGIIRT E: Bombesin, Vasopressin, and EndotheUn Rapidly Stimulate Tyrosine Phosphorylation in Intact Swiss 3T3 Cells. Proc
,Val/ Acad
Sci L’SA
1991. ESi577ii581.
Reports that neuropeptides acting stimulate grosine phosphor)lation of signal transduction.
through G.protein-coupled receptors and. hence. initiate a novel pathwa)
88:11986-i990. 39.
165
.
RO~ENGIIRT E: Galanin Stimulates Ca’+ Molnositol Phosphate Accumulation, and Clonal Small CeU Lung Cancer Cells. Cntrcc~ Res 1991, the lirst time.
JH: Purilication that Activate PhosphoUpase G, Class. J
Rozengurt
GL, BOYER JL, MORRIS AJ. HARDEN TK: Purification an AlF4 and G-protein py-Subunit-regulated Phospholipase C-activating Protein. / Biol Cbem 1991, 26614217-14225.
Nail
80:63X243X6. 30.
and cell proliferation
266:1820&18216.
of Mast Ceil Heparin Synthesis by the Rat Proc Nut/ Acrrd Sci USA 1991.
and Factor.
40.
factors
SMHCM AV. ~hYlJ:R JR, BROU,‘N KO, STI~KMVZIS PC: Regulation of Polyphosphoinositide-specfic Phospholipase C Activity by Purified Gq. Scimcr 1991, 251:804-807.
E Rozengun. Imperial Cancer Research Inn Fields. London WCM 3PX. UK.
Fund,
PO Box
123, 4r Lincoln’s
factors and cell proliferation Enrique
Imperial
Cancer
Rozengurt
Research
Fund, London,
UK
New advances in the identification of ligands and signal transduction events that induce cell proliferation through tyrosine kinase and C-protein-coupled receptors have extended our understanding of the pathways that control cell proliferation by growth factors. Current
Opinion
in Cell Biology,
Introduction
[I*] ). III every case. the formation of stable complexes requires the yrosine kinase activity of the receptor. The association of qtoplasmic proteins with the activated receptors is mediated by a non-catalytic domain of w 100 amino acids, the src homology (SH)Z region (revicared in [2-e] 1. These domains, expressed ;I$ bacterial fusion proteins, are sufficient to fomi stable complexes in lttt-0 with the liganded receptors [3*,9]. It has been proposed that vrosine l~tiospho~~lation of specific residues in the poljpeptide chain of the receptor acts as a switch to induce high-aJ%niv binding of SHZ-containing cytoplasmic proteins. Differences in the amino acid sequences between SH2 domains appear to result in distinct binding affinities [ 2**] Hence, SH2 domains plrly a crucial role in the association of q~oplasmic proteins with cellular and receptor h’rosine kinases.
The proliferation of most cells can he stimulated by ;t variety of extracellular ligands that act in a combinatorial and q~lcrgistic f&lion. These factors bind to receptors located on the cell surfrice and acti\.ate multiple signalling path\~iys. This re\,ie\v \vill focus on the earl!. c\‘ents in the action of gro\\Th factors. Lritcr steps in the cell cycle, in. eluding the role of 133-i~(‘~~aiid cyclins, will he the subject of 3 sepzirate section. At lczst two major signal-transduction l-xtth”‘a)~s initiate ca~~cades of molecular events leading to cell proliferation: one involves pol!peptide growth t:,ictors [e.g. pluteletderived growth ktc’tor ( PDGF) and epidermal growth Etctor ( EGF) ] that bind to receptors with intrinsic vrosine kinnse :lcti\it) :ind the other invohes receptors coupled by 8l[:liiine-nl[cIeoticle binding proteins (G proteins 1 to etfector activation. During the past year considerable Xl. vaiices have been made in the identifcation of extracelMar frictors :ind in the elucidation of signal-transduction c\‘ents that induc~e cell proliferation through both the t\‘rosinc kinase :md the G l”otein-mccliated path\~i!.s.
Tyrosine Signal
kinase
1992, 4:161-165
It is known that PDGF and EGF receptors associate with and phosphoqlatc PLC-y,, but the precise role of this moclifcrltion remains unclear. Recent evidence demonstr;ltes that PLC-), phosphorylation is directly responsible for the activation of this enqme in li\ring cells. Thus, o\~erexpression of PLC-y, and PLC-y1 increases tyrosine phosl~ho~lation of these enzymes and inositol phosphatc formation in response to PDGF [ 51. The mechanism of this acti~ltion was investigated by substituting phen!U:lnine for qnjsine at phosphoyl:ltion sites 77 1, ‘83 and 125-r, aiid expressing the mutant enqmes in 3T3 cells. Phen~~l:tlanine substitution ;Lt Tyr7H3 completely blocked the accumulation of inositol phosphates induced b!r PDGF 16**]. These results provide direct e\idence that the PDGF receptor stimulates the function of ;m intracellular signal-transducing protein by site-specilic n’rosine I~hosphon’lation.
receptors
transduction
There is consiclerablc e\~iclence th:it poI)~qXicle gro\\Th fnctors. including PDGF and EGF. activate their corresponding receptors 1~). inducing receptor dimerization and subsequent transl~hosl~ho~,t~itioi~ at specific vrosine residues. These liganclccl receptors ph!.sic:lll!. :wsociatc with mcl phosplloIyl~ltc :i set of q~op’lasmic proteins iniplic:itecl in intracellular sign:il transduction P;lth~~l)s. These include the y, isoform of I”)l}l’hosphoinositidespecifc l~hosl~holil~;is~ C ( PLC-y, ). l~‘hosl~1iatid!~lii~ositol 3’ kinase (Pl3Ii ). 1~21 U’S GTP;ise-;1cti\.;lting protein (GAP ). t)rrosinc kinases of the Src fiiniil)~ and other cellular proteins that have not. ;is !rct, been identified ( re\iciwecl in
The association
of Pl3K with PDGF receptors has continattention. This enzyme phosphotylates the inositol ring of I’hosptlLltidylinositol at the D3 position and is con~l~osed of t\vo subunits with apparent m&cular m:lsses of 85 and 1 10 kD [ 71. The 85 kD subunit has been cloned in Merent laboratories and shown to LIC’CI to ;lttl’act
Abbreviations BDNF-brain-derived
HCFphepatocyte PDCF-
growth
platelet-derived
neurotrophic factor; factor; NFl--neurofibromatosls growth
EGF
efxlermal growth factor; gene product; NCF-nerve
factor; Pl3K-phosphatldyllnosltol SCLC ~-small cell lung cancer;
@
Current
Biology
SH-src
Ltd
(;AP~~~CTPase-activating growth
3’kinase; PKC-protein homology domain.
ISSN
0955-0674
factor; kinase
protein; NT-3-neurotrophin-3;
C;
PL-phospholipase;
161
162
Cell multiplication
. contain SH2 domains that mediate the association of PI3K to phosphorylated receptors, but lacks catalytic activity [8*-lo=]. A synthetic peptide of 20 amino acids representing a conserved region of the kinase insert domain blocked the interaction in zpifro of the PI3K with the phosphorylated j%PDGF receptor [ 111. The precise biological significance of PI3K remains unknown. It has been thought, however, that it plays a role in eliciting cell proliferation, as deletion mutants in the p-PDGF receptors abolish both PI3K association and DNA .synthesis [lo]. In contrast, a deletion mutant of the a-PDGF receptor, lacking 80 amino acids of the kinase insert region, failed to show receptor-associated PI3K but retained partial mitogenic activity [ 121. Furthermore, mutations in the a-PDGF receptor that markedly impair its association with PUK did not prevent PDGF-induced mitogenesis [ 13**]. This demonstrates that the association of PI3K to a-PDGF receptor is not required for PDGF-induced mitogenic signalling. Future experiments should elucidate whether this constitutes a crucial difference in the mechanism of action of a- and P-PDGF receptors. The upstream and downstream mechanisms by which mitogenic stimuli engage ~21 rs in cellular signalling remain poorly understood. The findings that GAP associates with and serves as a substrate for phosphotylation by liganded tyrosine kinase receptors suggested a link between receptors and p2Ira mediated by GAP, but substantial evidence was lacking. It is known that phosphorylated GAP associates with two other cellular proteins, p62 and ~190. EGF treatment stimulates GAP phosphotylation and induces complex formation between GAP and ~190 [ 14.1. Interestingly, this complex has reduced GAP activity. This finding suggests that GAP phosphorylation plays a role in the regulation of p21raq activity. A region of the neurofibromatosis gene product (NFl) that has sequence similarity to the catalytic domain of GAP also stimulates p2 1 ra$ GTPase activity. Interestingly, NFI and GAP are regulated in l&-o by lipid mediators, particularly arachidonic and phosphatidic acid [ 15*,16]. Elucidation of the physiological significance of these effects in the regulation of p2I ra requires further experimental work. Ligands
for Trk, met and kit
During the past year, a number of ligand-receptor systems have been elucidated. The Trk family of tyrosine kinases has been identified as the cellular receptors for the neurotrophins, consisting of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3). The product of the proto-oncogene Trk serves as a high-affinity receptor for NGF [17-l whereas T&B, which exhibits a high degree of homology to Trk, has been identified as the receptor for BDNF [W-21*]. NT-3 can bind to both Trk and T&B, as well as to a novel member of the Trk receptor family, TrkC, that does not recognize either NGF or BDNF [ 22.1. These studies demonstrated that a previously identified NGFbinding protein, known as the low-affinity NGF receptor is not required for neurotrophin-mediated signal transduction. In common with other activated growth factor receptors, binding of NGF to Trk rapidly increases the phosphotylation of PLC-y in responsive cells [ 231. These
studies constitute an important step towards understanding the signal transduction pathways that mediate the biological effects of the neurotrophins. Another series of studies revealed a relationship between hepatocyte growth factor (HGF), scatter factor and the c-met proto-oncogene tyrosine kinase receptor. HGF is a potent mitogen for hepatocytes in culture and promotes liver regeneration after partial hepatectomy. HGF also stimulates the proliferation of endothelial and epithelial cells [ 241. Independently, an activity that stimulated the motility of certain epithelial cells (e.g. Madin-Darby canine kidney cell line) had been isolated and termed scatter factor (reviewed in [25*] 1. The sequences of cDNA clones encoding these polypeptides were virtually identical [26-l. Moreover, both purified HGF and scatter factor bind and stimulate tyrosine phosphotylation of c-nzef [26*,27*]. Thus, scatter factor and HGF are the same polypeptide and are the ligands for the c-met tyrosine kinase receptor that mediates the stimulation of cell motility or proliferation in the corresponding target cells. An important development has been the identification of the product of the Steel locus of the mouse as a growth factor. It was known that mutations in this loc~~saRect the development of a variety of cell lineages and that the Sfeel and the \V%ite Spotti,zg loci are distinct genes with closely related functions (reviewed in [ 281). The Steel growth factor has been purified from several sources and shown to stimulate the proliferation of mast [ 291 and hemopoietic cells [ 30). This novel growth factor has been identified as the ligand for the c-kit proto-oncogene receptor tyrosine kinase, which is encoded by the Wite Spolfiq locus. Addition of Steel growth factor to normal mast cells stimulates rapid tyrosine phosphorylation of c-kit and induces the association of PI3K and PLC-7, to the liganded receptor [31*].
G-protein-coupled Ligands
receptors
and receptors
An increasing number of neuropeptides and vasoactive peptides (e.g. bombesin, vasopressin, bradykinin, endothelin and thrombin) have been shown to act as potent cellular growth factors and have been implicated in a vdriety of normal and abnormal biological processes including development and tumorigenesis. During the past year, an important finding has been that the colony growth of small cell lung cancer (SCLC) cells is stimulated by multiple neuropeptides, including bombesin, bradykinin, cholecystokinin, neurotensin and vasopressin [32.*]. Further studies showed that galanin acts as a cellular growth factor for SCLC cells [ 33.1. These novel findings are important because they suggest that the previously identified autocrine growth loop of bombesin-like peptides is only one of an extensive network of paracrine and autocrine circuits that sustain the proliferation of SCLC. The cloning and sequencing of the cDNA for several mitogenic peptide receptors including bombesin [ 3d*], neuromedin-B [ 35.1, bradykinin [ 36.1, thrombin [37.01 and vasoactive intestinal peptide [38] has been accomplished during the past year. The fundamental struc-
Growth
tural feature of these G-protein-coupled receptors the seven putative transmembrane domains - has been found in every case. Nevertheless, the expression cloning of the thrombin receptor revealed a novel mechanism of receptor activation. The physiological &and of the receptor is generated by proteolytic cleavage of the aminoterminal extracellular domain of the receptor [37**]. It will be interesting to determine whether all biological effects of thrombin are initiated by the same receptor or by other receptor subtypes.
Signal transduction
Many neuropeptide receptors are known to rapidly stimulate the PLC-mediated hydrolysis of polyphosphoinositides, but the molecular identity of the phospholipases and G proteins involved remains elusive. Although there is direct evidence indicating that PLC-?/ is a target for growth factor receptors with intrinsic tyrosine kinase activity (see the preceding section), a different PLC isoform, namely PLC-p, has been shown to be regulated by G proteins. This PLC isoform does not contain SH2 domains. An important development in delineating this signal transduction pathway has been the purification of novel heterotrimeric G proteins of the Gq subfamily. When incorporated into phospholipid vesicles together with PLC-p, the guanine nucleotide regulation of this PLC isoform was reconstituted [3!+41]. A role for PLC-p and Gq in the mechanism of action of growth-promoting peptides is plausible, but definitive evidence is not yet available. In contrast, GTPase-inhibiting mutations of ai.2, which were found in certain endocrine turnours, have been shown to convert this pertussis toxin-sensitive G protein into a stimulator of cell proliferation (42.1. Elucidation of the precise mitogenic signal transduction pathway activated by this mutant G protein requires further experimental work. The activation of PLC-y and PLC-p (as well as that of phosphatidylcholine-specific PLC or PLD) by polypeptide growth factor and mitogenic peptides generates diacylglycerol, the second messenger that activates protein kinase C (PKC). Because the role of this kinase family in cell proliferation is well established, it is essential to identify and characterize its physiological substrates. An acidic, myristoylated protein that migrates with an apparent molecular mass of 80 kD has been identified as one of the most prominent substrates for PKC, both in cultured cells and in several tissues. Recently, the cDNAs encoding the substrates from rat brain [43*], Swiss 3T3 cells [44**], murine macrophage (451 and human brain [46] have been cloned. Comparison of the rodent sequences with those of the PKC substrates from human and bovine brain (termed MARCKS) revealed considerable sequence divergence, suggesting either a low level of evolutionary pressure or the existence of a gene family. Interestingly, PKC activation causes a dramatic down-regulation of the expression of mRNA and protein of the 8OkD substrate from Swiss 3T3 cells through a post-transcriptional mechanism [44**]. This novel finding suggests that this PKC substrate, which is a calmodulin and actin-binding protein, may play a suppressor role in the control of cell proliferation.
factors
and cell proliferation
Rozengurt
Activation of neuropeptide receptors linked to rapid polyphosphoinositide breakdown does not necessarily lead to identical long-term responses. Thus, bombesin induces a striking and sustained release of arachidonic acid and eicosanoid production in 3T3 cells, whereas vasopressin, bradykinin and endothelin fail to stimulate a similar response [47**]. Toxins have provided useful tools for dissecting signaltransduction pathways in eukaryotes. Mastoparan, a tetradecapeptide that directly stimulates G protein activity, induces arachidonic acid release and stimulates DNA synthesis in synergy with other factors in 3T3 cells [48*]. Pasteurellu multocida toxin, a 145kD protein, is a potent mitogen for cultured libroblasts. It induces striking polyphosphoinositide breakdown [49”] and constitutes the first intracellularly acting bacterial toxin that has been identified to activate this signal transduction pathway. Concluding
remarks
A striking feature of the diverse tyrosine kinase receptors summarized here is that in response to ligand binding (e.g. PDGF, EGF, libroblast growth factor, NGF, BDNF, NT-3 and, Steel growth factor), they rapidly associate with a specific set of SH2containing cytoplasmic proteins. An important challenge for the future is to determine whether the discrete number of cellular targets identified so far can account for all the molecular events (ionic fluxes, protein phosphotylation, gene expression etc) and biological responses induced by activation of these receptors. Classically, tyrosine phosphorylation by polypeptide growth factors and the generation of second messengers via neuropeptide receptors linked to G proteins have been regarded as separate though complementaty pathways in the mitogenic stimulation of cells. The recent demonstration that bombesin, vasopressin and endothelin stimulate tyrosine phosphorylation of specific substrates in Swiss 3T3 cells suggests the existence of an additional signal transduction pathway in the mitogenic response to these agents [50**]. Indeed, these peptides do not stimulate tyrosine phosphotylation of PLC-yt, PISK or GAP. Elucidation of the components of such a pathway, as well as the identity and function of the kinases and substrates, warrants further experimental work. References
and recommended
Papers of particular interest, published view, have been highlighted as: . of special interest .. of outstanding interest I. .
the annual
period
of re-
CNLEY LC, AUGER KR, CAHPENTEH C, Ducwotrnr B, GRAZ~ANI A, KAI’ELLER R, SOLTOFF S: Oncogenes and Signal Transduc-
tion. Detailed sociation
within
reading
Cell 1991, &281-302.
review on receptor tyrosine kinases and particularly on the asof wious q~oplasmic proteins with tyrosine kinase receptor.
Kocti CA, ANDEKON D, MORAN MF, Ettts C. PAWSON T: SH2 and SH3 Domains: Elements that Control Interactions of Cytoplasmic Signalling Roteins. Scicwce 1991, 252:66%674. in important review on the evidence for the role of SH2 domains in linking cellular proteins at receptor tyrosine kinases.
2. ..
163
164
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3. .
ANDERSON D, KOCH CA, G&7 I EUS C, Mot&v Binding of SH2 Domains of Phospholipase Src to Activated Growth Factor Receptors.
MF, PA~XON T: Cyl, GAP. and Scietzcc, 1990,
250:97%982. SH2 domains 4.
5.
can directly
bind
lig.mded
EGF
and PDGF
receptors.
MOHAMMADI M, HONEGGER AM, ROTIN D. FIXZHER R. BEILOT F. LJ W, DIONNE CA, JA~F M, RLIBINSTEIN M, SCIUSSINGER J: A Tyrosine-phosphorylated Carboxy-terminal Peptide of the Fibroblast Growth Factor Receptor (Fig) is a Binding Site for the SH2 Domain of Phospholipase C-yl. A/o/ Cc// Hiol 1991. 11:5068-5078. Su~liw.4~ I+ Euls C, LIN L-l PAWON T, KNOW J: Plateletderived Growth Factor Increases the In Vitro Activity of Phospholipase C-y, and Phospholipase C-y?. :I101 Cell Rio1 1991. 31:201~2025.
6. ..
K~hr HK, Klh! JW. ~ll~3~R5ll3N A, MAR R. 1151)&v J, Torn’ N. Shlllll AD, h1OR64\ SJ, COL~RTNEII~GE SA, PARKI~R PJ. WATE~I~X~) MJ: Characterization of Two 85 kd Proteins that Associate with Receptor Tyrosine Kinases, Middle-T/ppbOC-SrC Complexes. and PI 3. kinase. Cc// 1991. 65:91-IO+. This paper and [8*.9-l describe the purification and cloning through di&renr routes of the Hi kD subunit of P13K;. and drmonstnlte the presence of SH2 domains and binding of this protein IO liganded yrosine kinase receptors. 11.
12.
01-s~
THOhIP.sOI\I
ESCOHEDO JA, KAI~IIV\: DR, KA\;~~A~x;H WM. TI:RCK CW. WIUU!S LT: A Phosphatidylinositol-3 Kinase Binds fo Platelet-derived Growth Factor Receptors Through a Specific Receptor Sequence Containing Phosphovrosine. .1/o/ Cell Rio/ 1991, 11:1125-1132. HEIL>AKA~ MA. I’IERCE JH. lUhlBAKDl D, Rwc;~ew M. GUUXI JS, MATXII T, AARONSON SA: Deletion or Substitution Within the a Platelet-derived Growth Factor Receptor Kinase Insert Domain: Effects on Functional Coupling with Intracellular SignaIling Pathways. .1/o/ Cell Hiol 1991. 11:13+l-r2.
13. ..
Yll J-C, HEIIXRAN MA, PEIRCE JH, GL:XINI) JS, Lohle#.ni D. RLIGGI~RO M, AAKONSON SA: Tyrosine Mutations Within the a Platelet-derived Growth Factor Receptor Kinase Insert Domain Abrogate Receptor-associated Phosphatidylinositol3 Kinase Activity Without AITecting Mitogenic or Chemotactic Signal Transduction. ,llo/ Cell Biol 1331. 1 1:378C-3785. This paper suggese that Pl3K association wirh the a-PDGF receptor can be dissociated from mirogenesis. 13. .
MORAN IMF, POWS P, MCCO~L\IICK F, PAU’S~N T. EUJS C: Protein-tyrosine Kinases Regulate the Phosphorylation, Protein Interactions, Subcellular Distribution and Activity of ~21~ GTPase-activating Protein. ;Ilo/ Cc// Rio/ 1991, 11:1804-1812.
M: The Growth
65:89i-903. 19. .
10. .
E. BAIUACII) for Nerve
SOPPET D. EXXNIX)N E. MARN;OS J. MII~I)Il:h~~ DS, Rrw SW, BLAIR J, BLIRTON LE. STANTON BR. KAPIAN DR. IlwrxR T. NIKOIJCS K, I’Ak\l)r\ LF: The Neurotrophic Factors Brainderived Neurotrophic Factor and Neurotrophin-3 are Ligands for the lrkB Tyrosine Kinase Receptor. Cc>// 1991.
122-l.
9.
it.5 as
Goi.~wc M. TANAKA K. DOBROW~OLSKI S. WOOD D. Tw MH, MAR+LUL M. TA~MNOI F. STACEY DW: The GTPase Stimulatory Activities of the Neurolibromatosis Type 1 and the Yeast IRA2 Proteins are Inhibited by Arachidonic Acid. El/R0 ./ 1991. 10:298’-2903. Prwidrs elidewe that GAP and NFl are regulated itr lY/ro by lipid mcxiiators.
SW
.
is fo enhance
15. .
KR. COHEN B. and CharacterRat Liver. ./ Biol
7.
CAlU’ENlXR CL, DLIch~ORTH BC, AL’GER SCHAFFHAI!SE~ BS, CAXIUX LC: Purification ization of Phosphoinositide 3-kinase from C%rtn 1990, 32:1970-r-1971 1.
Emphasizes that the role ol’ GAF’ phosphorylation sociation with another cellular protein. ~190.
V. JINC S. L~w~hi~e F. T,~I.I~ P. BKYANI C, JONES KR. &ICHARI\KI)T LF. BruwcIi) M: Protein Kinase is a Receptor for BrainFactor and Neurotrophin-3. Cell 1991,
GWSS DJ. N~E SIH. I l.~~-LWPOltOs I’, MACC~II MJ. SQI’ISIO $I’. G7080. Reports the molecular cloning of the 80 kD PKC substrate from rat brain and reveals a novel post-translational modification.
Sc‘i 1 ‘SA 1901, 88:772+772H. Reports
Biof
43.
K: Expression
R\Y’. JAWAGIN
Receptor.
and Characterthe pl lsozyme C. Identifica-
d-3. .
rccepror.
35.
36.
A.
PETEKWN
of the Human /+DC N&l AC&
KE,
GOETXL
Vasoactive
.Sci LISA
EJ:
In1991,
Through a novel mt~hanism, Alsrelrre//~~ togen. is shown to induce the prtxluction identical to those induced by bombesin. 50. ..
multocidrc of inositol
toxin, a potent miphosphate species
%~L.WY I. GII. J. ~H%L+!!N W. SINNE’ITSMITH J. ROZENGIIRT E: Bombesin, Vasopressin, and EndotheUn Rapidly Stimulate Tyrosine Phosphorylation in Intact Swiss 3T3 Cells. Proc
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Reports that neuropeptides acting stimulate grosine phosphor)lation of signal transduction.
through G.protein-coupled receptors and. hence. initiate a novel pathwa)
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RO~ENGIIRT E: Galanin Stimulates Ca’+ Molnositol Phosphate Accumulation, and Clonal Small CeU Lung Cancer Cells. Cntrcc~ Res 1991, the lirst time.
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of Mast Ceil Heparin Synthesis by the Rat Proc Nut/ Acrrd Sci USA 1991.
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40.
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SMHCM AV. ~hYlJ:R JR, BROU,‘N KO, STI~KMVZIS PC: Regulation of Polyphosphoinositide-specfic Phospholipase C Activity by Purified Gq. Scimcr 1991, 251:804-807.
E Rozengun. Imperial Cancer Research Inn Fields. London WCM 3PX. UK.
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PO Box
123, 4r Lincoln’s
