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(Circulation Research. 2001;89:641.)
© 2001 American Heart Association, Inc.

Editorials

PI3King the L-Type Calcium Channel Activation Mechanism

Susan F. Steinberg

From the Departments of Pharmacology and Medicine, College of Physicians and Surgeons, Columbia University, New York, NY.

Correspondence to Susan F. Steinberg, MD, Associate Professor of Pharmacology and Medicine, Department of Pharmacology, College of Physicians and Surgeons, Columbia University, 630 W 168 St, New York, NY 10032. E-mail sfs1{at}columbia.edu

Key Words: PI3-kinase • calcium channels • G proteins • tyrosine kinases

In recent years, class I phosphoinositide 3-kinases (PI3Ks) have been implicated in an increasing number of signal transduction pathways linking virtually every class of extracellular stimulus to intracellular response.^1–3 Class I PI3Ks are enzymes that selectively phosphorylate the 3'-OH position of the PtdIns(4,5)P₂ inositol ring in vivo to generate PtdIns(3,4,5)P₃, which then can be further metabolized by inositol lipid phosphatases to PtdIns(3,4)P₂. PtdIns(3,4)P₂ and PtdIns(3,4,5)P₃ are nominally absent in resting cells, rise briskly in response to class I PI3K activation during cellular stimulation, and function in signal transduction and membrane trafficking largely as a result of their interaction with pleckstrin homology (PH) domains (100 amino acid 3-phosphoinositide binding modules) in a range of cellular proteins. Class I PI3Ks have been subclassified further according to their structure and mode of activation by cell surface receptors (Figure, panel A). Class I_A PI3Ks are heterodimers composed of a catalytic subunit (the ubiquitous p110, more tissue-restricted p110ß, or p110 which is confined to hematopoietic cells) tightly complexed to a regulatory adapter subunit (p85, p85ß, p55, or their splice variants). All catalytic subunits bind Ras, but the role of this interaction in PI3K signaling is uncertain. Regulatory subunits harbor a C-terminal p110-binding region flanked by two SH2 domains that dock the holoenzyme to the membrane through interactions with specific phosphotyrosyl-containing sequences within the C-terminus of receptor tyrosine kinases or other membrane-associated proteins. Larger p85 regulatory subunits also contain a series of N-terminal modular domains that specify other protein-protein interactions and dictate differences in holoenzyme targeting and regulation. Class I_B PI3K is composed of the p110 catalytic subunit (lacking the p85 subunit binding region) associated with a p101 regulatory protein. Class I_B PI3Ks are specifically stimulated by Gß dimers liberated upon G protein–coupled receptor (GPCR) activation. The p110 catalytic subunit contains all the structural elements necessary for Gß-induced stimulation; the p101 noncatalytic regulatory subunit differentially modulates its kinase activity. p101 supports p110 activation of c-Jun amino-terminal kinase, but has little effect on activation of extracellular signal–regulated protein kinase (ERK).^4,5 p101 alters the substrate preference of Gß-stimulated p110; p110 alone preferentially phosphorylates PtdIns to form PtdIns(3)P, whereas the p101/p110 heterodimer is a highly PtdIns(4,5)P₂-selective enzyme.⁶

View larger version (92K): [in this window] [in a new window]   A, Domain structure of class 1 PI3K isoforms. B, PDGF and angiotensin II receptors in rat protal vein myocytes couple to L-type calcium channel activation through pathways that involve distinct PI3K isoforms.

The convenient classification of class I PI3K isoforms according to their mechanism of activation has been challenged by recent evidence that PI3Kß is synergistically activated by GPCRs and receptor tyrosine kinases. p110ß catalytic subunits are directly activated by membrane-bound ß dimers; G subunits, tyrosine phosphorylation of p85 subunits, and p85 subunit interactions with phosphotyrosine-containing proteins are not required.⁷ However, like other class I_A enzymes, a phosphotyrosyl peptide interaction with the p85 regulatory subunit also leads to activation of PI3Kß. Hence, the p85/p110ß complex is a coincidence detector that integrates signals from GPCR- and tyrosine kinase–signaling cascades. Differential expression and regulation of PI3Kß and PI3K extends the repertoire and specificity of GPCR signaling to PI3K isoforms.

The best-characterized target for PI3K-derived lipid products is Akt, which is recruited to the plasma membrane through an interaction between its PH domain and 3'-phosphorylated phosphoinositides.² This promotes a conformational change that exposes Thr-308 and Ser-473 residues that then become phosphorylated by PDK1, resulting in full in vivo enzyme activation. Like Akt, PDK1 contains a PH domain that binds PtdIns(3,4)P₂ and PtdIns(3,4,5)P₃ with high affinity, ensuring PDK1 and Akt colocalization at specific membranes. Other targets for PtdIns(3,4,5)P₃-dependent phosphorylation by PDK1 include the activation loops of p70S6-kinase and protein kinase C (PKC) isoforms. PtdIns(3,4,5)P₃ binds to a PH domain in the nucleotide exchange factor Vav and allosterically enhances its exchange activity toward Rac, Cdc42, and RhoA, causes the PH domain-mediated membrane targeting of phospholipase C, and arguably competes with phosphotyrosine-containing proteins at the SH2 domains of Src and p85 regulatory subunits. Hence, PI3K enzymes initiate an intricate network of signals that influence cellular functions ranging from cell survival, proliferation, and migration to vesicular trafficking.

In addition to their function as lipid kinases, PI3K and PI3K both have intrinsic serine/threonine kinase activity. While most studies fail to consider this dual substrate specificity, p100 phosphorylates itself, its associated p85 regulatory subunit (which results in decreased lipid kinase activity for the complex), and the insulin receptor substrate-1.¹Inhibitors such as wortmannin and LY294002 cannot be used to discriminate PI3K lipid and protein kinase activity because they interfere with both. However, 3'-phosphorylated lipid-dependent signals can be identified with a PI3K mutant engineered to be selectively defective in lipid- (and not protein-) kinase activity. Studies with this mutant indicate that the protein kinase activity of PI3K is sufficient to activate the ERK cascade (although the direct target for protein phosphorylation has not been defined). In contrast, 3'-phosphorylated lipids are required for PI3K/Akt activation.⁸

Classical gene knockout strategies in the mouse have been used to examine PI3K isoform selective functions. The observations that targeted deletion of p110 is embryonic-lethal and that p110 null fibroblasts cannot be induced to proliferate in culture have been taken as evidence that p110 plays a fundamental role in cell proliferation.⁹ p110 null mice survive, but display a defect in inflammatory cell migration, abnormal T-cell function, and (according to one group of investigators) an increased incidence of colon tumors.^10,11 These distinct phenotypes are consistent with studies in cell culture identifying distinct roles for individual PI3K isoforms in mitotic signaling, cell migration, and cytoskeletal regulation.³

There is still relatively limited information on the role of individual PI3Ks in cardiovascular functions. A recent study reported that neonatal rat cardiomyocytes coexpress PI3K, ß, and , and that purinergic receptor stimulation with ATP results in the selective stimulation of PI3K (rather than PI3Kß), which plays a critical role in purinergic receptor regulation of spontaneous IP₃-induced calcium spiking. This occurs via a pathway involving PI3K and Fyn (a Src family kinase) activation, PIP₃-induced membrane anchoring of Tec (a Btk family member) and phospholipase C, and the concerted activation of phospholipase C by Tec and Fyn.¹² The role of PI3K in normal developmental growth also has been examined in the mouse heart. Expression of a constitutively active p85-p100 chimera during mouse heart development leads to increased heart (and cardiomyocyte) size, whereas dominant-negative p100 induces the reciprocal phenotype.¹³ Neither construct induces abnormalities in cardiac architecture/morphology or chamber function. The mechanism for PI3K action has been explored in neonatal cardiomyocyte cultures, where a PI3K-Akt-glycogen synthase kinase 3ß pathway is reported to transcriptionally activate atrial natriuretic factor¹⁴ and PI3K-Akt signaling is reported to induce cardioprotection during ischemic insults.^15,16

A study in this issue of Circulation Research (and a companion study in the Journal of Biological Chemistry) follows on a series of studies that examine the role of PI3K isoforms in receptor activation of voltage-gated L-type calcium channels in rat portal vein myocytes.^17,18 The major findings are that exogenous expression of all PI3K isoforms leads to increased L-type calcium channel activity, but channel activation by endogenous receptor tyrosine kinase and the GPCR agonists (platelet-derived growth factor [PDGF] and angiotensin II) follows distinct PI3K isoform-dependent pathways (Figure, panel B). L-type calcium channel activation by PDGF is mediated by PI3Kß. This pathway is defective in myocytes freshly isolated from portal veins (which express p110 and p110, but not p110ß) and is reconstituted with cell culture and the induction of endogenous p110ß expression. This and previous studies identify a different pathway for L-type calcium channel activation by the GPCR agonist angiotensin II that involves ß₁₃ dimers (released from G₁₃), PI3K, and PKC.^18–20 It is surprising that exogenous p110 supports phosphotyrosyl peptide-dependent calcium channel activation, but endogenous p110 expression fails to link functional PDGF or angiotensin II receptors to calcium channel activation. Angiotensin II receptors are devoid of endogenous tyrosine kinase activity, but numerous studies identify angiotensin II activation of Src (or transactivation of epidermal growth factor receptors) in aortic vascular tissues. In theory, these mechanisms could support PI3K activation, but differences in angiotensin II receptor signaling to tyrosine kinase pathways between vascular beds may explain the PI3K selectivity observed in these studies. The failure of PDGF to activate PI3K is not readily attributable to differences in known upstream signaling molecules. Indeed, the experiments by Macrez et al¹⁷ do not distinguish whether the lesion is in PDGF activation of endogenous PI3K or in endogenous PI3K stimulation of the calcium channel. In either case, the results are consistent with previous evidence that the structurally homologous p110 and p110ß catalytic subunits perform unique functions, and that changes in p110ß expression and availability calibrate PI3K-dependent signaling.⁷ The mechanism for calcium channel activation by PI3K isoforms also remains uncertain, as several (nonexclusive) mechanisms are possible, including direct effects of PtdIns(3,4,5)P₃ to modulate channel gating, PtdIns(3,4,5)P₃-dependent activation of PDK1 and phosphorylation/activation of a PKC isoform, or an analogous pathway leading to the activation of Akt (particularly because Akt has been implicated in L-type calcium channel activation in neurons²¹). Finally, there is no reason to exclude the PI3K protein kinase activity as an additional mechanism that might regulate calcium channel activity.

One of the more interesting mechanistic questions raised by this study is how different extracellular stimuli specifically signal through distinct PI3-kinase isoforms. One obvious potential mechanism is differential compartmentalization of receptors, PI3-kinase isoforms, and/or channels to membrane subdomains. This mechanism is appealing, given recent literature describing lipid rafts (membrane subdomains particularly enriched in phosphoinositide lipids) as nucleation centers for signaling pathways. Although not reported for calcium channels, differential partitioning of potassium channels to lipid raft microdomains has been proposed as a mechanism to ensure efficient regulation by kinases and phosphatases.²² The presence of three distinct class I_A p110 catalytic subunits that form complexes with any of seven distinct adapter proteins provides an additional mechanism for specificity through independently regulatable cassettes that link tyrosine kinases and PI3K signaling. The versatility of the regulatory subunit N-terminal modular domain structure in dictating the protein composition (and hence the regulation and function) of the holoenzyme is emphasized by recent evidence that only PI3K holoenzymes with the long form of the regulatory subunit regulate the actin cytoskeleton; the p85 N-terminal motifs are not required for DNA synthesis (a less spatially organized cellular process²³). Future research will undoubtedly disclose yet additional mechanisms that increase the repertoire of signaling specificity for PI3K enzymes. The application of this information to studies of L-type calcium channel regulation in the heart and vasculature is a challenge, as these are technically demanding studies. However, insofar as L-type calcium channels represent the major pathway for calcium entry during excitation-contraction coupling in cardiac and vascular myocytes, meeting this challenge may lead to fundamental insights into cardiac regulatory controls and identify novel drug targets.

Acknowledgments

This work was supported by United States Public Health Service–National Heart, Lung, and Blood Institute grants HL-28958 and 64639.

Footnotes

The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.

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