In the present study, we provided evidence that RACK1 regulates directed cell migration, and it does so through a novel mechanism that works on the Gβγ and effector interface. Our data indicate that RACK1 acts as a negative regulator of chemotaxis. Thus, silencing RACK1 enhances SDF1α- and fMLP-stimulated chemotaxis of Jurkat and dHL60 cells, whereas overexpressing RACK1 abrogates cell migration. The fact that RACK1 inhibits chemotaxis of both Jurkat and dHL60 cells induced via two different GPCRs suggests that RACK1 may have a general role in regulating GPCR-mediated leukocyte migration.
Several lines of evidence indicate that RACK1 regulates GPCR-directed cell migration by acting on Gβγ to intervene selectively in the activation of its effectors PLC and PI3K. First, the activity of PLC and PI3K can be significantly augmented by down-regulation of RACK1, whereas overexpression of RACK1 has an opposite effect. Second, inhibiting RACK1 neither affects Gα13-mediated RhoA activation, nor has an effect on Gβγ-mediated ERK activation, indicating that RACK1 selectively regulates Gβγ effectors. Third, the inhibitory effect of RACK1 on effector activation and chemotaxis can only be mimicked by its mutant BD1-2, which retains Gβγ-binding capacity but does not bind other RACK1-interacting proteins such as Src and PKCβ that are known to be involved in cell migration, suggesting that RACK1 exerts its function by physical association with Gβγ. Supporting this, endogenous RACK1 was found to form a complex with Gβγ, and these proteins are colocalized at the leading edge of both polarized Jurkat and dHL60 cells. Finally, our in vitro data provided direct evidence that RACK1 inhibits Gβγ-stimulated PLCβ and PI3Kγ activity by binding to Gβγ. We showed previously that RACK1 inhibits PLCβ activation by competing for its binding to the signal transfer region of Gβγ (Chen et al., 2005
). In the present studies, we demonstrate that RACK1 uses a similar mechanism to regulate Gβγ-stimulate PI3Kγ activation. Thus, as with PLCβ2
, RACK1 and its mutant BD1–2 inhibit PI3Kγ in a dose-dependent manner. Compared with GRK2ct, RACK1 displays a lower potency in inhibiting PI3Kγ (IC50
~ 20 μM vs. 4 μM for GRK2). Mathematical simulation indicates that the lower potency of RACK1 in inhibiting PI3Kγ reflects the relatively lower binding affinity of Gβγ to RACK1 (EC50
~ 500 nM) than GRK2ct (EC50
~ 100 nM; data not shown; Pumiglia et al., 1995
; Chen et al., 2005
). It is worth noting that we do not know the exact binding affinity of RACK1 to Gβγ in intact cells because RACK1 may undergo posttranslational modification, which could significantly alter its binding ability. However, the relatively lower potency of RACK1 in inhibiting Gβγ signaling may facilitate its ability to regulate Gβγ effectors in a spatiotemporal-specific manner because the inhibitory effect of RACK1 will critically depend on its relative local concentration in cells to the effectors.
Using a peptide-based approach, we also identified residues on Gβγ that are critical for PI3Kγ activation. Although in this assay the concentration of these peptides required to inhibit PI3K is relatively high and the effect of inhibition (15–40%) is modest, we believe that this reflects the relative contribution of the corresponding residues of the peptides to Gβγ-mediated PI3K activation rather than nonspecific effects. First, the inhibitory effect of the peptides is effector-specific. For example, in our previous studies (Chen et al., 2005
), the peptide p44–54 inhibits the binding of Gβγ to PLCβ2 by 60% and directly activates PLCβ2, but it does not affect either the basal or Gβγ-stimulated PI3K activity in the current studies. Similarly, although the peptide p177–189 caused more than 70% inhibition of Gβγ-dependent PLCβ2 activity (Chen et al., 2005
), it does not affect PI3K activation. Conversely, the peptide 309–316, which inhibited PI3K activation by 20%, had no effects on PLCβ2 activation (Chen et al., 2005
). Second, some of the Gβγ effector-binding sites identified by the peptide-based approaches have been corroborated by site-directed mutagenesis studies. For example, by substituting the charged amino acid within residues 44–54 of Gβ with alanines, we confirmed the results from the peptide-based studies that these residues play a critical role in PLCβ2 activation (Chen et al., 2005
). Although we have not yet evaluated the contribution of different amino acids of Gβ to PI3K activation, previous studies from Garrison's group demonstrated that mutation of residues H311, R314, and V315 impaired the ability of Gβγ to activate PI3Kγ (Kerchner et al., 2004
). These residues are contained in the peptide 309–316, which inhibited ~20% of Gβγ-dependent PI3K activity in our studies. Though our data remain to be corroborated by a high-resolution crystal structure of the Gβγ/PI3Kγ complex, our findings that some of Gβ residues (residues 86–105 and 309–316) critical for PI3Kγ activation are contained in the RACK1 contact surface on Gβγ is consistent with the notion that RACK1 regulates cell migration by physical association with and steric hindrance of the access of specific effectors to Gβγ.
Our findings that RACK1 regulates chemotaxis via PLCβ and PI3Kγ are consistent with the roles of these two enzymes in leukocyte migration, as demonstrated by numerous studies from gene knockout mice of PLCβ2/3
and PI3Kγ and with pharmacological inhibitors (Rickert et al., 2000
; Hawkins et al., 2006
). However, the relative contribution of these two pathways in RACK1-mediated regulation of chemotaxis remains to be elucidated.
Like Gβ, RACK1 has numerous interacting proteins and is generally considered as a scaffold/adaptor protein involved in diverse of cellular processes. It has also been shown to be involved in migration of epithelia cells, partially through its interaction with PKCβ or PKCε, Src, and integrins (Buensuceso et al., 2001
; McCahill et al., 2002
; Buensuceso et al., 2005
; Kiely et al., 2005
; Doan and Huttenlocher, 2007
). However, conflicting reports of RACK1 in either promoting or inhibiting cell migration are found in literature, probably reflecting the context-dependent functions of RACK1 in cells, tissues, and stimuli (Buensuceso et al., 2001
; Doan and Huttenlocher, 2007
). In our studies, we did not detect interaction of RACK1 with either PKCβ or Src in Jurkat cells. Moreover, in the transwell assay, Jurkat cell migration through the filter membrane does not rely on cell adhesion to extracellular substrates via integrins. These data suggest that RACK1 is unlikely to modulate leukocyte migration through its effect on the activity of PKCβ, Src, or integrins. Supporting this, the RACK1 mutant BD1–2, which does not bind PKCβ and Src, can still mimic the inhibitory effect of the full-length RACK1. Moreover, studies with pharmacological inhibitors of Src, PKCβ/ε, and ERKs indicate that they are not involved in SDF1α-stimulated Jurkat cell migration.
Our data unambiguously demonstrate that RACK1 plays a negative role in regulating leukocyte migration. Moreover, they provide further support for our previous notion that RACK1 can tune Gβγ activation of effectors to impact specific functions of Gβγ (Chen et al., 2004a
). This mechanism of modulating cell migration is different from that used by other known regulators. For example, the phosphatases PTEN and SHIP impinge on directional sensing and motility of cells by dephosphorylating PIP3
(Franca-Koh et al., 2007
; Nishio et al., 2007
). Moreover, they are essential for the establishment of internal gradient of signaling molecules and directional sensing of Dictyostelium
and neutrophils (Funamoto et al., 2002
; Iijima and Devreotes, 2002
; Li et al., 2005
; Nishio et al., 2007
). In contrast, RACK1 is not essential for gradient sensing as inhibition of RACK1 neither affects the translocation of PH-AKT-GFP to the leading edge nor the directional migration of dHL60 cells. Unlike RACK1, other regulatory proteins such as arrestins and GRKs negatively modulate chemotactic responses of leukocytes by down-regulating functions of chemokine receptors through phosphorylation and internalization of GPCRs (Vroon et al., 2006
). Regulator of G protein signaling (RGS) proteins inhibit chemotaxis by shortening the lifetime of the active Gα-GTP subunit (Kehrl, 2006
). Given the fact that RACK1 modulates cell migration via competitive inhibition of Gβγ effector activation, it is conceivable that dynamic changes in the expression level of RACK1 during physiological and pathological processes of immune responses may contribute to fine tuning of leukocyte function. Additionally, because RACK1 is ubiquitously expressed, and chemokine receptors are involved in the migration of diverse cell types, including fibroblasts and endothelial and tumor cells, RACK1 may also function in other cellular processes, such as wound healing, angiogenesis, and tumor metastasis (Gillitzer and Goebeler, 2001
; Balkwill, 2004
; Strieter et al., 2005