Calmodulin Activates Phosphatidylinositol 3-Kinase*

Calmodulin and phosphatidylinositol 3-kinase are vital components of a number of common intracellular events. Calmodulin, a ubiquitous Ca2+-dependent effector protein, regulates multiple processes in eukaryotic cells, including cytoskeletal organization, vesicular trafficking, and mitogenesis. Phosphatidylinositol 3-kinase participates in events downstream of the receptors for insulin and other growth factors. Here we demonstrate by coimmunoprecipitation and affinity chromatography that Ca2+/calmodulin associates with Src homology 2 domains in the 85-kDa regulatory subunit of phosphatidylinositol 3-kinase, thereby significantly enhancing phosphatidylinositol 3-kinase activity in vitro and in intact cells. Furthermore, CGS9343B, a calmodulin antagonist, inhibited basal and Ca2+-stimulated phosphorylation of phosphatidylinositol in intact cells. These data demonstrate a novel mechanism for modulating phosphatidylinositol 3-kinase and provide a direct link between components of two fundamental signaling pathways.

kinase and CaM are common components of several fundamental intracellular processes. For example, wortmannin, an inhibitor of PI3-kinase, and CGS9343B impede insulin-induced glucose uptake (3)(4)(5). Both CaM (6,7) and PI3-kinase (8) participate in early endosome fusion. In the cytoskeleton, PI3kinase has been linked to actin rearrangement (9), binds ␣-, ␤and ␥-tubulin (10), and plays a role in platelet-derived growth factor-and insulin-induced membrane ruffling (11,12). Overexpression of CaM alters cell morphology and the arrangement of microfilaments within the cell (13). In addition, CaM binds to a variety of cytoskeletal proteins (1) including the family of unconventional myosins (14) and has been implicated in osteoclast membrane ruffling (15) and the formation of microspikes in neuronal cells (16). Finally, both PI3-kinase (17) and CaM, via modulation of the association of IQGAP1 with Cdc42 (18), may participate in the regulation of Rho family GTPases. Since CaM and PI3-kinase modulate similar cellular events, we evaluated a possible interaction between these two signaling components.

EXPERIMENTAL PROCEDURES
Cell Culture and Lysis-Sf9 cells were maintained in Grace's medium supplemented with 10% fetal bovine serum and infected with baculovirus as described previously (19). CHO cells were grown to 80% confluence in Ham's F-12 medium with 10% fetal bovine serum. 32D cells and 32D cells expressing rat IRS-1 (32D/IRS-1) were cultured as described previously (20). The medium was removed, cells were washed 3 times with phosphate-buffered saline, and 1 ml of lysis buffer (50 mM Tris base, pH 7.4, 150 mM NaCl, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 0.1 g/ml leupeptin, aprotonin, and pepstatin, and 1% Triton X-100) was added. Where indicated CaCl 2 or EGTA was included. The cells were collected and quick-frozen in methanol/dry CO 2 .
Antibodies-The specific anti-calmodulin monoclonal antibody has been previously described (21). The anti-myoglobin monoclonal antibody (IgG 1 ) was highly purified by Dr. J. Ladenson (Washington University Medical Center, St. Louis). The anti-p85 antibody was prepared by immunizing rabbits with a glutathione S-transferase (GST) fusion protein containing the inter-SH2 region of p85. The anti-p85 monoclonal and the anti-GST and anti-p110 polyclonal antibodies were purchased from Upstate Biotechnology Inc.
CaM-Sepharose-Cell lysates were incubated for 2 h at 4°C with 20 l of CaM-Sepharose or Sepharose alone in the presence of 0.1 mM CaCl 2 or 1 mM EGTA. GST fusion proteins were incubated with CaM-Sepharose for 1 h at 25°C in the presence of 0.1 mM CaCl 2 or 1 mM EGTA. Samples were washed 5 times in lysis buffer containing 0.1 mM CaCl 2 or 1 mM EGTA, resolved by SDS-PAGE, and immunoblotted as described below.
Immunoprecipitation and Immunoblotting-Equal amounts of protein lysate were immunoprecipitated with either anti-CaM monoclonal antibody, anti-myoglobin monoclonal antibody, anti-p85 antibody, or preimmune serum. Samples were washed five times in lysis buffer, resolved by SDS-PAGE, and transferred to PVDF, and immunoblots were probed with anti-p85, anti-p110, or anti-CaM antibody. Complexes were visualized with the appropriate horseradish peroxidase-conjugated secondary antibody and developed by ECL.
PI3-kinase Activity-Anti-CaM immunoprecipitates, anti-p85 immunoprecipitates, or CaM-Sepharose beads were resuspended in 15 mM Hepes, pH 7.4, 5 mM MgCl 2 , 0.3 mM EGTA, 0.24 mg/ml phosphatidylinositol, and 20 M [␥-32 P]ATP in a final volume of 100 l. In selected experiments, 0.1 mM CaCl 2 (EGTA was omitted), 0.5, 2, or 5 M CaM, 100 M LY294002, or 100 M pY608 were added singly or in combination as indicated in the figure legends. The reaction was stopped with 100 l of 1 M HCl, and 200 l of 1:1 CHCl 3 :methanol was added. After centrifugation, the lower phase was spotted onto Silica Gel 60 plates, and thin-layer chromatography was performed using CHCl 3 /methanol/ NH 4 OH/dH 2 O (45:35:1.5:8.5) as the solvent. Where indicated, phosphatidylinositol phosphate (PtdIns-P) was located by autoradiography and excised, and 32 P was determined by liquid scintillation counting. For HPLC analysis, PtdIns-P was extracted from the silica gel, deacyl-* This work was supported in part by National Institutes of Health Grants DK43682, DK47308, and DK09062 and by the JDF. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

RESULTS AND DISCUSSION
The 85-kDa regulatory subunit of PI3-kinase (p85) was precipitated from lysates of baculovirus-infected Sf9 cells expressing p85 with CaM-Sepharose but not with Sepharose alone, demonstrating an interaction between these two proteins (Fig.  1A). To identify the CaM-binding region of p85, CaM-Sepharose was incubated with GST fusion proteins containing various regions of p85. Probing with anti-GST antibody disclosed that the carboxyl-terminal SH2 domain bound to the CaM-Sepharose (Fig. 1B). Longer exposure of the blot revealed association of the amino-terminal SH2 domain with CaM-Sepharose, suggesting that this region also interacts with CaM albeit with a lower affinity (data not shown). Binding to either SH2 domain was significantly reduced when Ca 2ϩ was chelated with EGTA (data not shown). Furthermore, endogenous p85 from CHO cell lysates bound to CaM-Sepharose only in the presence of Ca 2ϩ (Fig. 1C). No binding of CaM to the SH3 or breakpoint cluster homology regions was detected.
Insulin activates PI3-kinase by inducing the association of the SH2 domains of p85 with specific phosphotyrosine-containing motifs of IRS proteins (19). It has been demonstrated that a phosphorylated YMXM peptide (pY608), derived from amino acids 605-615 of IRS-1, activates PI3-kinase by binding to its SH2 domains (23). Incubation with pY608, but not with the corresponding nonphosphorylated peptide (Y608), resulted in displacement of baculovirus-expressed p85 from CaM-Sepharose (Fig. 1D). These data suggest that CaM binds in or near the pocket occupied by phosphorylated YMXM motifs. Among the functional possibilities that may be inferred from this observation are that CaM may directly activate PI3-kinase or may modulate PI3-kinase activity by competing with tyrosinephosphorylated proteins for binding to the SH2 domains of p85.
To resolve this question, the PI3-kinase activity associated with CaM was examined. The binding of PI3-kinase from CHO cell lysates to CaM-Sepharose was Ca 2ϩ -dependent ( Fig. 2A). Anti-CaM and anti-p85 antibodies immunoprecipitated p85 and p110, the catalytic subunit of PI3-kinase, from CHO cell lysates (Fig. 2B); however, p85 was not coimmunoprecipitated with an irrelevant isotype-identical monoclonal antibody (antimyoglobin) or preimmune serum (Fig. 2B). Even though more p85 and p110 were present in anti-p85 immunoprecipitates than in anti-CaM immunoprecipitates, the PI3-kinase activity in the anti-CaM immunoprecipitates was 6-fold greater than that in anti-p85 immunoprecipitates (Fig. 2C). These data, coupled with the relatively small amount of catalytic p110 subunit bound to CaM, strongly support the contention that CaM significantly stimulates PI3-kinase activity. HPLC analysis of the PtdIns-P produced by PI3-kinase in the anti-CaM immunoprecipitate revealed that 80% was phosphatidylinositol 3-phosphate (PtdIns-3-P) (Fig. 2D). Greater than 95% of the phosphatidylinositol kinase activity in CHO cell lysates was phosphatidylinositol 4-kinase (data not shown), supporting a specific interaction of CaM and PI3-kinase. Furthermore, the PI3-kinase inhibitor LY294002 (24) decreased the PI3-kinase activity in the anti-CaM immunoprecipitates by greater than 90% (Fig. 2E), further substantiating the identity of this enzyme as PI3-kinase.
Since CaM binds to IRS-1 in a Ca 2ϩ -sensitive manner (25), the possibility of a ternary complex between CaM, p85, and IRS-1 was evaluated in 32D cells, which lack endogenous IRS-1 and IRS-2 (26). Essentially identical amounts of p85 from ly- sates of 32D and 32D/IRS-1 cells were precipitated with CaM-Sepharose (Fig. 3), indicating that the binding is independent of IRS proteins.
To further confirm our results, activation of PI3-kinase by pY608 or CaM was compared using anti-p85 immunoprecipitates of CHO cell lysates. In anti-p85 immunoprecipitates, 5 M CaM stimulated PI3-kinase activity by 50%, while 100 M pY608 enhanced PI3-kinase activity by 38% (Fig. 4). EGTA did not significantly alter basal or pY608-stimulated PI3-kinase activity in the anti-p85 immunoprecipitates (data not shown) but abolished the activation of PI3-kinase by CaM (Fig. 4). These data verify that CaM stimulated PI3-kinase in a Ca 2ϩdependent manner. The activity of baculovirus-expressed PI3-kinase was similarly augmented by CaM (data not shown). Thus, the activation of PI3-kinase by CaM was comparable to that obtained by occupancy of the SH2 domains with a phosphopeptide, implying that the binding of CaM to the SH2 domains of p85 is a novel mechanism for the regulation of PI3-kinase activity.
To examine whether Ca 2ϩ /CaM activates PI3-kinase in situ, [Ca 2ϩ ] i was increased with the ionophore A23187 (25) in CHO cells preloaded with 32 P. 32 P-Labeled phospholipids were then extracted and resolved by TLC, and phosphatidylinositol phosphates were excised and examined by HPLC. Treatment with A23187 increased PtdIns-3-P by 30% (Table I) prevented the stimulation by increased [Ca 2ϩ ] i . A23187 induced the formation of phosphatidylinositol 3,4-diphosphate (PtdIns-3,4-P 2 ) (which was not detected in lipids extracted from control cells), and this effect was abrogated by concomitant incubation with CGS9343B. Phosphatidylinositol 3,4,5trisphosphate (PtdIns-3,4,5-P 3 ), the predominant phospholipid produced in response to insulin and other growth factors (2, 27, 28), was not detected under any conditions examined. The production of phosphatidylinositol 4-phosphate (PtdIns-4-P), but not phosphatidylinositol 4,5-diphosphate (PtdIns-4,5-P 2 ), was also enhanced by increased [Ca 2ϩ ] i and was sensitive to CGS9343B (Table I). The CaM-sensitive PI4-kinase activity may be mediated by a mammalian CaM-stimulated inositol trisphosphate 4-kinase similar to that identified in plants (29).
This study establishes a novel direct interaction between Ca 2ϩ /CaM and the SH2 domains of p85 resulting in the activation of PI3-kinase. Increased [Ca 2ϩ ] i , acting through CaM, modulates PI3-kinase activity in intact cells. Interestingly, the specific phosphatidylinositol phosphates generated by Ca 2ϩ / CaM differ from those induced by incubating cells with insulin or growth factors (2,27,28). We demonstrate that changes in [Ca 2ϩ ] i regulate PI3-kinase in a manner distinct from the canonical phosphotyrosine-dependent pathway, providing an additional level of control of this fundamental enzyme. Our findings expand the repertoire of enzymes that are regulated by Ca 2ϩ /CaM and accentuate the myriad interconnections between intracellular signaling pathways.