Inositol phospholipid-specific phospholipase C: interaction of the γ isoform with tyrosine kinase

TIBS 16 - AUGUST 1991

THE BINDING of CaZ'-mobilizing growth factors, neurotransmitters and hormones to their corresponding receptors triggers a series of events that leads to the activation of phospholipase C (PLC), also known as phosphoinositidase. Activated PLC hydrolyses inositol phospholipids to generate inositol phosphates and diacylglyerol, which serve as intraceilular second messengers~. Evidence accumulated over the past several years from direct protein isolation, immunological characterization and molecular cloning studies suggest that there are four types of PLC in mammalian tissues 0x, 15,7 and 8) all of which are single poiypeptides, the products of discrete genes2,3.Molecular masses measured by polyacrylamide gel electrophoresis (SDS-PAGE) are 62-68kDa for PLC-a, 150-154kDa for PLC-13, 145-148kDa for PLC-7 and 85-88 kDa for PLC-6 (Ref. 2). All four enzymes have similar catalytic properties: they hydrolyse three common phosphoinositides, phosphatidylinositol (PI), phosphatidylinositoi 4-phosphate (PIP) and phosphatidylinositoi 4,5-bisphosphate (PIPz), and their catalytic activities are dependent on the concentration of Ca2" with the hydrolysis of PIP2being most sensitive. Initially, cDNAs corresponding to the PLC~, -7 and -8 from rat or bovine brain were cloned and sequenced4-7. Comparison of the deduced amino acid sequences of these cDNAs revealed a significant sequence similarity in two regions, one of approximately 150 amino acids and the other of approximately 240 amino acids. The two similar domains, designated X and Y in Fig. 1, were about 60% and 40% identical, respectively, between the three PLCs. Preceding the X domain in each of the three enzymes is a region of about 300 amino acid residues with a sequence identity of only 20%. Although the sequence of PLC-a has been established 8, it was not included in Fig. 1 as its authenticity is debatable: the cDNAs for PLC-IS, -7 and -6 have been expressed in heterologous cells, with a resultant increase in cellular PLC activRy, whereas similar expression stud-

Inositol phospholipid-specific phospholipase C interaction of the "/isoforn? with tyrosine kinase f

The generation of second messengers from inositol phospholipids is catalysed by enzymes from the phospholipase C family. Activation of phospholipase C-¥z through tyrosine phosphorylation provides a link between mitogenic and inositol phospholipid signaling. ies have not been performed with the putative PLC-a cDNA. Recently, four new PLC-related cDNAs were cloned and sequenced 9.1°. Sequence alignment has shown that each of the new sequences is similar to and structurally related to one of the

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three brain PLCs previously isolated, rather than a new type of PLC. This suggests that there is more than one enzyme of each PLC type; subscript arabic numerals are used to designate the members of each type, with the three enzymes isolated from rat or bovine

43%

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© 1991,ElsevierSciencePublishers, (UK) 0376-5067/91/$02.00

297

TIBS 16 - AUGUST 1991

brain named PLC-IS~, -7~ and -81. The Drosophila PLC gene it, norp A, most

closely resembles the PLC-[5 type (Fig. I). For members of the same type of PLC, the X and Y domains are the most highly conserved, but there is also significant homology outside these domains. For example, PLC-[3~ and -[32 exhibit 54% identity in the amino-terminal region preceding X and 31% identity iT, the carboxy-terminal region following Y (see Fig. I). The members of a particular PLC type are highly conserved in different tissues and species. For example, amino acid conservation is greater than 95% for PLC-7~ from rat, bovine and human brain, and PLC-7~ appears to be expressed in almost every mammalian tissue. In a previous review ~, we proposed a fifth type of PLC, PLC-e; however, it now appears appropriate to consider PLC-~ (85 kl)a) as a member of the PLC-~type. Structural

and

functional domains of PLC-7

PLC-y~ and -~2 have high sequence identities in the amino-terminal 300 amino acids and in the region of ,tOO amino acids between domains X and Y. In the latter region, PLC-y~ and -Y2contain three domains that are related in sequence to two limited portions ol the product of the src oncogene known as SH2 and SH3 (src homology 2 and 3)4,5. SH2 and SH3 were first recognized as highly conserved regions in the regulatory domains of a number of nonreceptor tyrosine kinases, such as the products of abl and src (the catalytic region of these src-related tyrosine kinases is named SHI) ~. PLC-yt can therefore be structurally divided into three domains: X, Y and SH (SH2 plus SH3). The function of each domain has been studied by expressing various plasmids encoding truncated versions of PLC-7 in Escherichia coil (PLC-yz) 9 or COS-I cells (PLC-7~)~°. PLC activity was s~ill clearly detected when the SH domain was deleted, although reduced to 10-307/o of the control. Deletion of either the X or Y domain led to complete loss of activity. It appears, therefore, that X and Y, but not the SH domain, are essential for PLC activity. A diverse family of proteins that do not exhibit kinase activity also contain either SH2 or SH3, or both (Fig. 2). These ;~roteins include the product of the gab~rh oncogene of CTI0 virus~3; the GTPase-activating proteiF of ras p2i (GAP)~4; myosin-IB of Acanthamoe~S; the yeast actin-binding protein, ABPI~6; the CDC25 gene product, which regu-

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fates the ras and adenylate cyclase pathway in yeast=6; the FUS-I product, which is required for cell fusion during yeast conjugatio@; non-erythrold 0¢-spectrin~e; and two neutrophll cytosolic factors, NCF47K and NCF65KiT, which are required for activation of NADPH oxldase. SH2 and SH3 can be located anywhere in the primary structure of these proteins and can appear together (e.g. PLC-7,the src product) or separately (e.g. myosin-IB, CDC25). Furthermore, the number of SH domains is variable: PLC-7~and GAP contain two SH2 domains, whereas NCF65K and NCF4;'K contain two SH3 domains. These results suggest that SH2 and SH3 domains have independent functions. Although the deduced amino acid sequence of PLC-Tt provides little information concerning the precise role of SH2 and SH3 domains in transmembrahe signaling, the structural relations to the above-mentioned proteins may provide important clues. For example, the crk protein, which consists almost entirely of SH2 and SH3 domains, associates with a variety of phosphotyrosine-containing proteins including p60v~, but not with tyrosine phosphatefree forms of these proteins ~3. Matsuda et al. 13therefore proposed that the role

of SH2 and SH3 domains is to modulate protein-protein interactions in response to tyrosine phosphorylation. Recently, a phosphotyroslne-recognizing function for SH2 was suggested; bacterially expressed $H2 domains of PLC-Tz~4, GAP~4 and the abl tyroslne klnase ~s bound tightly to a number of tyrosinephosphorylated proteins, including activated receptors for epidermal growth factor (EGF) and plateletderived growth factor (PDGF). The SH3 domain appears independently of SH2 in several proteins that have no apparent functional similarity (Fig. 2). Drubin et al. ~e noticed that a common feature of SH3-containing proteins is physical or functional association with the cortical actin cytoskeleton, a submembranous protein network that is important for the regulation of cell shape, cell adherence and cell movement. ABPI, myosin-l, spectrin and several src-related proteins are all associated with the membrane cytoskeleton ~. Deletion within the SH3 region of p60v-~ affects the ability of the protein to associate with the detergent-insoluble cytoskeletal matrix. An ATP-insensitive actin-binding site has been mapped to the carboxy-terminal 250 amino acid residues of myosin-l, in

TIBS 16 - AUGUST 1991

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Themechanism of activation of PLC-¥1by EGF or PDGF.(a) Binding of EGF (PDGF)to its receptor elicits autophosphorylation. (b) The PLC-y1

forms a complex with the EGFphosphorylated (PDGF)receptor and the tyrosine residues 771, 783 and 1284 are phosphofflated, whichactivates PLC-¥1,(¢).

which the SH3 domain is located TM. It is also significant that brush-border myosin-I lacks an SH3 domain and does not contain an ATP-insensitive actinbinding site. Furthermore, both NCF47K and NCF65K translocate from cytosol to the membrane skeleton with activation of the oxidative burst in phagocytic cells ~T.

with EGF or PDGF causes a tight association of PLC-7~ with the corresponding growth factor, such that the receptor and PLC-¥~can both be precipitated by an antibody to either PLC-7~ or the receptor ~9-z4. Purified EGF and PDGF receptors phosphorylate PLC-7~ at the same sites as those that are phosphorylated in cells (i.e. tyrosine residues 771, 783 and 1254)2sa°. The role of tyrosine phosphoryl-tion was investigated by Activationof PLCfflthroughtyroslne substituting phenylalanine for tyrosine phosphorylatlon The biological actions of PDGF and at these three sites of PLC-Ts and EGF are mediated through cell-surface expressing the mutant enzymes in NIH transmembrane receptors that possess 3T3 cells 27. Phenylalanine substitution ligand-activated cytoplasmic tyrosine at Tyr783 completely blocked the actikinase activity. The early cellular vation oi PLC by PDGF, whereas muevents induced by the binding of these tation at Tyr1254 inhibited the growth factors to their respective response by 40% and mutation at receptors include receptor autophos- Tyr771 enhanced the response by 50%. phorylation at several tyrosine residues The decreased response to PDGF of the and the stimulation of PLC activity. cells harboring mutant PLC-7~ was not These events appear to require the because the phenylalanine substitution intrinsic tyrosine kinase activity of the at tyrosines inactivated the enzymes. receptors since mutant PDGF and EGF Mutation at residues 771, 783 or 1254 receptors that lack tyrosine kinase did not affect the catalytic activity of activity bind the growth factors but fail PLC-7~measured in vitro zT. Like the wildtype enzyme, PLC-7~ substituted with to stimulate the hydrolysis of PIP2. Recently we have shown that treat- phenylalanine at Tyr783 became associment of a number of cells with EGF or ated with the PDGF receptor and was PDGF increases both the tyrosine and phosphorylated at serine residues in serine phosphate content of PLC-7~,but response to PDGF. These results sugnot of PLC-JS]or PLC-5~19-24,and that the gest that PLC-7~ is the isozyme of PLC tyrosine phosphorylation of PLC-7~ cor- that mediates PDGF-induced inositol relates well with the increased turnover phospholipid hydrolysis and that phosof PIPz24. In addition, treatment of cells phorylation on Tyr783 is essential for

PLC-7~ activation. Furthermore, they show that neither the association of PLC-7~ with the PDGF receptor nor its phosphorylation on serine residues is sufficient to account for PDGF-induced activation of PLC-7~. As yet, the significance of serine phosphorylation of PLC-Tsis unknown. A plausible mechanism by which EGF (or PDGF) activates PLC-7~ is shown in Fig. 3. In unstimulated cells, most PLC-7~ (>95%) is present in the cytosol. Binding of EGF to the extracellular domain of its receptor elicits the phosphorylation of several tyrosine residues at its carboxy-terminal regionz8 (Fig. 3a). These phosphotyrosine residues are recognized by th,~ SH2 domains of PLC-7~,leading to formation of the coimmunoprecipitable receptor-PLC-7~ complex and phosphorylation of PLC-7~ tyrosine residues 771, 783 and 1254 by the receptor kinase domain (Fig. 3b). This model is consistent with the recent observation that SH2-containing proteins bind a truncated carboxyterminal region of the EGF receptor containing all known tyrosine autophosphorylat~on sites in a tyrosine phosphorylation-dependent manner 29. The receptor-PLC-7~ association could be further strengthened by interaction between the receptor tyrosine kinase domain and tyrosine residues 771, 783 and 1254 of PLC-7~.Short synthetic peptides containing the individual tyrosine

299

TIBS 16 - AUGUST 1991

phosphorylation sites of PLC-y~ exhibited binding affinities (Kin) of -0.4 mM for the autophosphorylated EGF receptor kinase3°; this result predicts that the phosphorylation of PLC-7~ by the EGF receptor might take place, albeit slowly, even in the absence of the SH2 (PLC-7])-phosphotyrosine (EGF receptor) association, unless a conformational change induced by this association is necessary to expose the three phosphorylation sites on PLC-T~. Thus, the phosphotyrosine content of PLC-y~associated with the receptor is significantly less than that of unassociated PLC-~,~,suggesting that after phosphorylation of the three tyrosine residues, PLC-Iftis rapidly released from the receptor. The model shown in Fig. 3c assumes that the two SH2 domains in the tyrosine-phosphorylated PLC-7~, after release of the enzyme from the EGF receptor, interact intramolecularly with the PLC-7~ tyrosine phosphates. However, there is no direct evidence supporting such an intramolecular interaction. In the model, phosphotyrosines 783 and 1254 are chosen as the SH2-binding sites because full activation of PLC-~,~ in NIH 3T3 cells by PDGF required phosphorylation at tyrosine 783 and 1254 but not phosphorylation at Tyr771 (Ref. 27). The intramolecular interaction is presumed, in the model, to elicit a conformational change that allows the SH3 domain to bind to the membrane cytoskeleton and brings the putative catalytic domains X and Y to the cytoplasmic face of the cell membrane where the PLC substrate PIP~ is located. This model is consistent with the proposed role of the SH3 domain TM and with the observation that EGF or PDGF treatment of cells causes translocation of PLC-7~from a predominantly cytosolic localization to membrane fractions 3|,3s. The catalytic activity of PLC-yI measured with exogenous substrates is not affected by tyrosine phosphorylation. Furthermore, overproduction of PLC-7~in NIH 3T3 cells did not affect the background level of PIP2 hydrolysis in the absence of PDGF, despite the fact that PLC activity in cell homogenates was proportional to the extent of expression. However, overexpression of PLC-~,~ correlated with PDGF-induced hydrolysis of PIP~. These results suggest that PLC-~,~activity in intact cells may be tightly regulated by a negative modulator, candidates for which include proteins with molecular sizes of I00, 84 and 47 kDa that are coimmuno-

300

precipitated with PLC-T~ by antibodies Activation of PLC-~,by non-receptortyrosine to PLC-T119's].Whether any of these pro- kinases teins actually plays a role in the modulation of PLC-T] activity is unknown. Additional candidates for the negative modulator are proteins that associate with PIP2. For example, profilin, a cytosolic actin-binding protein, binds four or five molecules of PiPs with high affinity and inhibits hydrolysis of this lipid by unphosphorylated PLC-T~, but not by PLC-T~phosphorylated by purified EGF receptor kinase~3. Recently, glucosphingolipid was suggested as a negative modulator of PLC because cells depleted of their glucosphingolipid exhibited elevated PLC activity34. Two putative inhibitors, a PLC-interacting protein inhibitor and a PiPs-interacting inhibitor, are depicted in Fig. 3a and 3c, respectively. In the absence of these negative modulators, we would not expect to be able to measure any change in activity of PLC-T~as a result of phosphorylation. Recently, Triton X-100 was shown to selectively inhibit the PIPs-hydrolysing activity of unphosphorylated PLC-T~, mimicking the role of profilin or glucosphingolipid35. Therefore, the growth factordependent activation of PLC-T~is likely achieved by eliminating the inhibitory effect of negative modulation. If this is the case, our data indicate that phosphorylation of Tyr783 is critical for the elimination of inhibition and that phosphorylation of Tyr1254 is required for complete elimination, whereas phosphorylation of Tyr771 is not necessary for relief from inhibition. Although it is evident that the EGF receptor activates PLC-y~ through tyrosine phosphorylation, a guanine nucleotide-binding protein (G protein) also appears to participate in the EGFdependent hydrolysis of inositol phospholipids in certain cells: EGF-mediated inositol phosphate accumulation in rat hepatocytes 3°and renal epithelial cellsa7 is inhibited by pertussis toxin. However, in other cell types, notably murine epithelial A-431 cells, 3T3 fibroblasts, vascular smooth muscle ceils and liver epithelial WB cells, the e{fects of EGF and PDGF are not attenuated by pertussis toxin. It thus appears that different cell types may utilize different mechanisms for coupling the EGF receptor to activation of PLC. Possible roles for a G protein include augmentation of the tyrosine phosphorylation-dependent activation of PLC-TI and activation of a PLC other than PLC-~/~.

Growth factors such as EGF, PDGF and fibroblast growth factor (FGF) activate PLC-¥]through tyrosine phosphor.j!ation and the receptors for these growth factors are protein tyrosine kinases. However, ligation of membrane IgM in B lymphocytes and the multicomponent T cell receptor-CD3 complex ~CR-CD3) in T cells causes activation of non-receptor protein tyrosine kinases and of PLC. Recent data suggest that tyrosine phosphorylation of PLC-¥: is responsible for the activation of PLC in B cells3sand T cells39,despite the fact that neither membrane lgM nor any of the TCR-CD3 components is a protein tyrosine kinase. This result suggests that certain receptors are capable of recruiting a soluble tyrosine kinase and activating PLC-~/: by a mechanism similar to that described for the EGFreceptor.

Activation of PLC-~/2 PLC-¥s also contains SH2 and SH3 domains. Whereas the equivalents of tyrosine residues 771 and 783 of PLC-¥~ are conserved in PLC-lfs, an equivalent of Tyr1254 is not present in PLC-¥s. Therefore, one can speculate that the activation of PLC-¥2involves interaction with a phosphotyrosine-containing protein, probably a protein tyrosine kinase with a substrate specificity different from those that phosphorylate PLC-¥:. The nature of the protein tyrosine kinase and extracellular signals linked to the activation of PLC-lf2are not known.

G protein regulation of PLC In addition to tyrosine phosphorylation-dependent activation, a variety of extracellular signals have been shown to regulate PLC activity through G proteins. There is much evidence suggesting that two types of G protein, pertussis toxin-sensitive and -insensitive G proteins, are involved in the PLC activation. The pertussis toxin-sensitive G protein has been purified from bovine brain 4° and bovine liver4~ and, on an immunologic basis, is a member of the recently described Gq class 42. This Gq protein specifically activates the [5~ isozyme but has no effect upon the ¥~ and 8~ isozymes4L The Gq family is comprised of several members with similar primary structure 42. It is therefore likely that PLC-J32is also activated by one of the Gq family members. Thus, the important remaining task is to find out whether PLC-5 is activated through a pertussis toxin-sensitive G protein.