Early Steps in Autophagy Depend on Direct Phosphorylation of Atg9 by the Atg1 Kinase

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An Atg1/Atg13 Complex with Multiple Roles in TOR-mediated Autophagy Regulation

The TOR kinases are conserved negative regulators of autophagy in response to nutrient conditions, but the signaling mechanisms are poorly understood. Here we describe a complex containing the protein kinase Atg1 and the phosphoprotein Atg13 that functions as a critical component of this regulation in Drosophila. We show that knockout of Atg1 or Atg13 results in a similar, selective defect in autophagy in response to TOR inactivation. Atg1 physically interacts with TOR and Atg13 in vivo, and both Atg1 and Atg13 are phosphorylated in a nutrient-, TOR- and Atg1 kinase-dependent manner. In contrast to yeast, phosphorylation of Atg13 is greatest under autophagic conditions and does not preclude Atg1-Atg13 association. Atg13 stimulates both the autophagic activity of Atg1 and its inhibition of cell growth and TOR signaling, in part by disrupting the normal trafficking of TOR. In contrast to the effects of normal Atg13 levels, increased expression of Atg13 inhibits autophagosome expansion and recruitment of Atg8/LC3, potentially by decreasing the stability of Atg1 and facilitating its inhibitory phosphorylation by TOR. Atg1-Atg13 complexes thus function at multiple levels to mediate and adjust nutrient-dependent autophagic signaling.     

Autophosphorylation Within the Atg1 Activation Loop Is Required for Both Kinase Activity and the Induction of Autophagy in Saccharomyces cerevisiae

Autophagy is an evolutionarily conserved degradative pathway that has been implicated in a number of physiological events important for human health. This process was originally identified as a response to nutrient deprivation and is thought to serve in a recycling capacity during periods of nutritional stress. Autophagy activity appears to be highly regulated and multiple signaling pathways are known to target a complex of proteins that contains the Atg1 protein kinase. The data here extend these observations and identify a particular phosphorylation event on Atg1 as a potential control point within the autophagy pathway in Saccharomyces cerevisiae. This phosphorylation occurs at a threonine residue, T226, within the Atg1 activation loop that is conserved in all Atg1 orthologs. Replacing this threonine with a nonphosphorylatable residue resulted in a loss of Atg1 protein kinase activity and a failure to induce autophagy. This phosphorylation required the presence of a functional Atg1 kinase domain and two known regulators of Atg1 activity, Atg13 and Atg17. Interestingly, the levels of this modification were found to increase dramatically upon exposure to conditions that induce autophagy. In addition, T226 phosphorylation was associated with an autophosphorylated form of Atg1 that was found specifically in cells undergoing the autophagy process. In all, these data suggest that autophosphorylation within the Atg1 activation loop may represent a point of regulatory control for this degradative process.MACROAUTOPHAGY (hereafter referred to as autophagy) is a highly conserved process of self-degradation that is essential for cell survival during periods of nutrient limitation (Tsukada and Ohsumi 1993). During autophagy, a double membrane grows out from a specific nucleation site, known as the pre-autophagosomal structure, or PAS, in Saccharomyces cerevisiae and the phagophore assembly site in mammals (Suzuki and Ohsumi 2007). This membrane encapsulates bulk protein and other constituents of the cytoplasm and ultimately targets this material to the vacuole/lysosome for degradation (Xie and Klionsky 2007). Recent studies have linked this pathway to a number of processes important for human health, including tumor suppression, innate immunity, and neurological disorders, like Huntington''s disease (Rubinsztein et al. 2007; Levine and Kroemer 2008). Determining how this pathway is regulated is therefore important for our understanding of these processes and our attempts to manipulate autophagy in clinically beneficial ways.Most of the molecular components of the autophagy pathway were initially characterized in the budding yeast, S. cerevisiae, but orthologs of many of these Atg proteins have since been found in other eukaryotes (Tsukada and Ohsumi 1993; Meijer et al. 2007). A complex of proteins that contains the Atg1 protein kinase is of special interest and appears to be a key point of regulatory control within this pathway (Kamada et al. 2000; Budovskaya et al. 2005; He and Klionsky 2009; Stephan et al. 2009). In S. cerevisiae, genetic and biochemical data indicate that this complex is targeted by at least three different signaling pathways. Two of these pathways, involving the Tor and cAMP-dependent protein kinases, inhibit this process, whereas the AMP-activated protein kinase is needed for the full induction of autophagy (Noda and Ohsumi 1998; Wang et al. 2001; Budovskaya et al. 2004; Stephan and Herman 2006; Kamada et al. 2010). The manner in which these signaling pathways regulate Atg1 activity and the precise role of this kinase in the autophagy process are presently matters of intense scrutiny.Although Atg1 kinase activity is required for the induction of autophagy, relatively little is known about how this enzyme is regulated in vivo. Two proteins associated with Atg1, Atg13 and Atg17, have been shown to be required for full Atg1 kinase activity both in vitro and in vivo (Kamada et al. 2000; Stephan et al. 2009). The roles of these proteins appear to be conserved through evolution as functional homologs of both have been identified in fruit flies and/or mammals (Hara et al. 2008; Chan et al. 2009; Chang and Neufeld 2009; Ganley et al. 2009; Hosokawa et al. 2009; Jung et al. 2009; Mercer et al. 2009). However, it is not yet clear precisely how these proteins stimulate Atg1 activity. In this study, we show that Atg1 is autophosphorylated within the activation loop and that this phosphorylation is required for both Atg1 kinase activity and the induction of autophagy. The activation loop is a structurally conserved element within the kinase domain and phosphorylation within this loop is often a necessary prerequisite for efficient substrate binding and/or phosphotransfer in the catalytic site (Johnson et al. 1996; Nolen et al. 2004). This loop generally corresponds to the sequence between two signature elements within the core kinase domain, the DFG and APE motifs (Hanks and Hunter 1995). Phosphorylation within this loop tends to result in a more ordered structure for this region and the proper positioning of key elements within the catalytic core of the kinase domain (Knighton et al. 1991; Johnson and O''reilly 1996; Huse and Kuriyan 2002). We found that Atg1 activation loop phosphorylation was correlated with the onset of autophagy and that replacing the site of phosphorylation with a phosphomimetic residue led to constitutive Atg1 autophosphorylation in vivo. In all, the data here suggest that Atg1 phosphorylation within its activation loop may be an important point of regulation within the autophagy pathway and models that discuss these data are presented.     

The Autophagy-related Protein Kinase Atg1 Interacts with the Ubiquitin-like Protein Atg8 via the Atg8 Family Interacting Motif to Facilitate Autophagosome Formation

In autophagy, a cup-shaped membrane called the isolation membrane is formed, expanded, and sealed to complete a double membrane-bound vesicle called the autophagosome that encapsulates cellular constituents to be transported to and degraded in the lysosome/vacuole. The formation of the autophagosome requires autophagy-related (Atg) proteins. Atg8 is a ubiquitin-like protein that localizes to the isolation membrane; a subpopulation of this protein remains inside the autophagosome and is transported to the lysosome/vacuole. In the budding yeast Saccharomyces cerevisiae, Atg1 is a serine/threonine kinase that functions in the initial step of autophagosome formation and is also efficiently transported to the vacuole via autophagy. Here, we explore the mechanism and significance of this autophagic transport of Atg1. In selective types of autophagy, receptor proteins recognize degradation targets and also interact with Atg8, via the Atg8 family interacting motif (AIM), to link the targets to the isolation membrane. We find that Atg1 contains an AIM and directly interacts with Atg8. Mutations in the AIM disrupt this interaction and abolish vacuolar transport of Atg1. These results suggest that Atg1 associates with the isolation membrane by binding to Atg8, resulting in its incorporation into the autophagosome. We also show that mutations in the Atg1 AIM cause a significant defect in autophagy, without affecting the functions of Atg1 implicated in triggering autophagosome formation. We propose that in addition to its essential function in the initial stage, Atg1 also associates with the isolation membrane to promote its maturation into the autophagosome.     

Contribution of Atg1-Dependent Autophagy to TOR-Mediated Cell Growth and Survival

The Ser/Thr kinase Atg1 (Ulk1/Unc51) appears to act as a convergence point for multiple signals that regulate autophagy, and in turn interacts with a large number of autophagy-related (Atg) proteins. Working in the Drosophila system, we recently found that overexpression of Atg1 is sufficient to induce autophagy, independent of upstream nutrient signals. We exploited this finding to examine the roles of autophagy in cell growth and death, and to test the interaction of Atg1 with the TOR signaling pathway. These studies provided genetic evidence that autophagy is a potent inhibitor of cell growth, and that high levels of autophagy lead to caspase-dependent apoptotic cell death in vivo. Atg1 also has an inhibitory effect on TOR signaling, indicating the existence of a positive feedback mechanism that may amplify the nutrient-dependent signals that control autophagy.

Addendum to:

Direct Induction of Autophagy by Atg1 Inhibits Cell Growth and Induces Apoptotic Cell Death

R.C. Scott, G. Juhász and T.P. Neufeld

Curr Biol 2007; 17:1-11     

MST Kinases Monitor Actin Cytoskeletal Integrity and Signal via c-Jun N-Terminal Kinase Stress-Activated Kinase To Regulate p21Waf1/Cip1 Stability

As well as providing a structural framework, the actin cytoskeleton plays integral roles in cell death, survival, and proliferation. The disruption of the actin cytoskeleton results in the activation of the c-Jun N-terminal kinase (JNK) stress-activated protein kinase (SAPK) pathway; however, the sensor of actin integrity that couples to the JNK pathway has not been characterized in mammalian cells. We now report that the mammalian Ste20-like (MST) kinases mediate the activation of the JNK pathway in response to the disruption of the actin cytoskeleton. One consequence of actin disruption is the JNK-mediated stabilization of p21^Waf1/Cip1 (p21) via the phosphorylation of Thr57. The expression of MST1 or MST2 was sufficient to stabilize p21 in a JNK- and Thr57-dependent manner, while the stabilization of p21 by actin disruption required MST activity. These data indicate that, in addition to being components of the Salvador-Warts-Hippo tumor suppressor network and binding partners of c-Raf and the RASSF1A tumor suppressor, MST kinases serve to monitor cytoskeletal integrity and couple via the JNK SAPK pathway to the regulation of a key cell cycle regulatory protein.The actin cytoskeleton is a dynamic structure that determines cell morphology and motility. In addition, the cytoskeleton also influences other biological functions, such as proliferation, survival, and death, although the mechanistic details linking the cytoskeleton to these processes have not been fully elucidated. Considerable effort has focused on characterizing the signal transduction pathways that control cytoskeletal organization (33). The actin cytoskeleton itself also may regulate cell signaling; for example, mechanical stretching, shear stress, and cytoskeletal disruption each have been shown to activate stress-activated protein kinase (SAPK) pathways (34). Although in Saccharomyces cerevisiae an actin integrity-responsive pathway has been identified in which actin cytoskeleton disassembly results in the activation of the Ssk2p kinase that lies upstream of the Hog1 SAPK pathway (7, 56), an analogous pathway in mammalian cells has not been delineated.SAPK pathways are specific examples of mitogen-activated protein kinase (MAPK) cascades (43). At the bottom of archetypal MAPK pathways are signal-propagating kinases such as ERK1 and ERK2; in the case of SAPK signaling, the similarly positioned kinases are JNK and p38 family members. MAPK are phosphorylated and regulated by MAPK kinases (MAP2K); for c-Jun N-terminal kinase (JNK), the MAP2K are MKK4 and MKK7, while for p38 they are MKK3 and MKK6. Moving stepwise further upstream are MAP3K and MAP4K, although in some pathways there may be no need for a MAP4K, the Ras activation of the MAP3K Raf in the ERK MAPK pathway being one example.Although much recent interest has focused on their antiproliferative and proapoptotic functions as a component of the Salvador-Warts-Hippo tumor suppressor network (31) and as binding partners of the c-Raf MAP3K (42) and RASSF1A tumor suppressor (39), the mammalian Ste20-like kinases 1 and 2 (MST1 and MST2, respectively) were first identified (17) because of their homology with the Saccharomyces cerevisiae Ste20 MAP4K that acts upstream of three MAPK cascades, including the Ste11/Pbs2/Hog1 SAPK pathway (51). Although the MST kinase domains are very similar to those in Ste20 and mammalian p21-activated kinases (PAK), there is little homology outside this domain, and as a result MST1 and MST2 make up their own Ste20 subfamily without direct orthologues prior to the emergence of the bilaterian subregnum. Given the homology with Ste20, initial characterization focused on the possibility that MST kinases were involved in MAPK regulation, and indeed MST kinases were found to activate SAPK pathways (27), which was associated with the activation of MKK6 and MKK7 (27). It also was found that MST1 coexpression with a kinase-dead version of the MAP3K MEKK1 blocked JNK activation (26). Consistently with these results, MST1 could not activate JNK in cells deleted for both MAP2K enzymes MKK4 and MKK7 (53). Therefore, it appears that MST kinases work at the same level (MAP4K) as Ste20 in the regulation of the SAPK pathways. Although proapoptotic signaling has been shown to contribute to MST activation via caspase-mediated proteolysis, which removes an autoinhibitory domain (27), little is known about how other nonapoptotic stimuli regulate MST.There are several possible consequences resulting from the activation of SAPK pathways in response to modifications to actin cytoskeleton organization or integrity. Actin disruption and consequent JNK activation may induce cell cycle arrest (23) or apoptosis (11), or it may promote cell survival (2). We previously showed that one way JNK activation following cytoskeletal disruption might contribute to cell cycle arrest is through the stabilization of the cyclin-dependent kinase inhibitor (CDKI) p21^Waf1/Cip1 (p21) (14). The eventual outcome of SAPK activation following actin cytoskeleton modification may be influenced by signal intensity, duration, and cellular context. Further progress toward determining how cytoskeletal disruption generates these outcomes will be possible when the details describing how actin cytoskeletal changes activate SAPK signaling have been established.We wished to determine whether MST kinases sense the integrity of the actin cytoskeleton and link with SAPK signaling. We found that MST2 was colocalized with filamentous actin structures. The expression of MST1 or MST2 was sufficient to activate JNK1, and cytoskeletal disruption activated MST as well as JNK1 in an MST-dependent manner. One consequence of actin disruption is the JNK-mediated stabilization of p21, which was determined to be via phosphorylation of Thr57. The expression of MST1 or MST2 was sufficient to stabilize p21 in a JNK- and Thr57-dependent manner, while the stabilization of p21 by actin disruption required MST activity. These data indicate that MST kinases serve to monitor cytoskeletal integrity and couple via the JNK SAPK pathway to the regulation of a key cell cycle regulatory protein.     

Role of Membrane Association and Atg14-Dependent Phosphorylation in Beclin-1-Mediated Autophagy

During autophagy, a double membrane envelops cellular material for trafficking to the lysosome. Human beclin-1 and its yeast homologue, Atg6/Vps30, are scaffold proteins bound in a lipid kinase complex with multiple cellular functions, including autophagy. Several different Atg6 complexes exist, with an autophagy-specific form containing Atg14. However, the roles of Atg14 and beclin-1 in the activation of this complex remain unclear. We here addressed the mechanism of beclin-1 complex activation and reveal two critical steps in this pathway. First, we identified a unique domain in beclin-1, conserved in the yeast homologue Atg6, which is involved in membrane association and, unexpectedly, controls autophagosome size and number in yeast. Second, we demonstrated that human Atg14 is critical in controlling an autophagy-dependent phosphorylation of beclin-1. We map these novel phosphorylation sites to serines 90 and 93 and demonstrate that phosphorylation at these sites is necessary for maximal autophagy. These results help clarify the mechanism of beclin-1 and Atg14 during autophagy.     

Nrbf2 Protein Suppresses Autophagy by Modulating Atg14L Protein-containing Beclin 1-Vps34 Complex Architecture and Reducing Intracellular Phosphatidylinositol-3 Phosphate Levels

Autophagy is a tightly regulated lysosomal degradation pathway for maintaining cellular homeostasis and responding to stresses. Beclin 1 and its interacting proteins, including the class III phosphatidylinositol-3 kinase Vps34, play crucial roles in autophagy regulation in mammals. We identified nuclear receptor binding factor 2 (Nrbf2) as a Beclin 1-interacting protein from Becn1^−/−;Becn1-EGFP/+ mouse liver and brain. We also found that Nrbf2-Beclin 1 interaction required the N terminus of Nrbf2. We next used the human retinal pigment epithelial cell line RPE-1 as a model system and showed that transiently knocking down Nrbf2 by siRNA increased autophagic flux under both nutrient-rich and starvation conditions. To investigate the mechanism by which Nrbf2 regulates autophagy, we demonstrated that Nrbf2 interacted and colocalized with Atg14L, suggesting that Nrbf2 is a component of the Atg14L-containing Beclin 1-Vps34 complex. Moreover, ectopically expressed Nrbf2 formed cytosolic puncta that were positive for isolation membrane markers. These results suggest that Nrbf2 is involved in autophagosome biogenesis. Furthermore, we showed that Nrbf2 deficiency led to increased intracellular phosphatidylinositol-3 phosphate levels and diminished Atg14L-Vps34/Vps15 interactions, suggesting that Nrbf2-mediated Atg14L-Vps34/Vps15 interactions likely inhibit Vps34 activity. Therefore, we propose that Nrbf2 may interact with the Atg14L-containing Beclin 1-Vps34 protein complex to modulate protein-protein interactions within the complex, leading to suppression of Vps34 activity, autophagosome biogenesis, and autophagic flux. This work reveals a novel aspect of the intricate mechanism for the Beclin 1-Vps34 protein-protein interaction network to achieve precise control of autophagy.     

Protein Kinase D2 Assembles a Multiprotein Complex at the Trans-Golgi Network to Regulate Matrix Metalloproteinase Secretion

Vesicle formation and fission are tightly regulated at the trans-Golgi network (TGN) during constitutive secretion. Two major protein families regulate these processes: members of the adenosyl-ribosylation factor family of small G-proteins (ARFs) and the protein kinase D (PKD) family of serine/threonine kinases. The functional relationship between these two key regulators of protein transport from the TGN so far is elusive. We here demonstrate the assembly of a novel functional protein complex at the TGN and its key members: cytosolic PKD2 binds ARF-like GTPase (ARL1) and shuttles ARL1 to the TGN. ARL1, in turn, localizes Arfaptin2 to the TGN. At the TGN, where PKD2 interacts with active ARF1, PKD2, and ARL1 are required for the assembly of a complex comprising of ARF1 and Arfaptin2 leading to secretion of matrix metalloproteinase-2 and -7. In conclusion, our data indicate that PKD2 is a core factor in the formation of this multiprotein complex at the TGN that controls constitutive secretion of matrix metalloproteinase cargo.     

LKB1 Represses Focal Adhesion Kinase (FAK) Signaling via a FAK-LKB1 Complex to Regulate FAK Site Maturation and Directional Persistence

Liver kinase β1 (LKB1, also known as STK11) is a serine/threonine kinase that has multiple cellular functions including the regulation of cell polarity and motility. Murine proteomic studies show that LKB1 loss causes aberrant adhesion signaling; however, the mechanistic underpinnings of this relationship are unknown. We show that cells stably depleted of LKB1 or its co-activator STRADα have increased phosphorylation of focal adhesion kinase (FAK) at Tyr³⁹⁷/Tyr⁸⁶¹ and enhanced adhesion to fibronectin. LKB1 associates in a complex with FAK and LKB1 accumulation at the cellular leading edge is mutually excluded from regions of activated Tyr³⁹⁷-FAK. LKB1-compromised cells lack directional persistence compared with wild-type cells, but this is restored through subsequent pharmacological FAK inhibition or depletion, showing that cell directionality is mediated through LKB1-FAK signaling. Live cell confocal imaging reveals that LKB1-compromised cells lack normal FAK site maturation and turnover, suggesting that defects in adhesion and directional persistence are caused by aberrant adhesion dynamics. Furthermore, re-expression of full-length wild-type or the LKB1 N-terminal domain repressed FAK activity, whereas the kinase domain or C-terminal domain alone did not, indicating that FAK suppression is potentially regulated through the LKB1 N-terminal domain. Based upon these results, we conclude that LKB1 serves as a FAK repressor to stabilize focal adhesion sites, and when LKB1 function is compromised, aberrant FAK signaling ensues, resulting in rapid FAK site maturation and poor directional persistence.     

Differential Involvement of Atg16L1 in Crohn Disease and Canonical Autophagy: ANALYSIS OF THE ORGANIZATION OF THE Atg16L1 COMPLEX IN FIBROBLASTS*

A single nucleotide polymorphism in Atg16L1, an autophagy-related gene (ATG), is a risk factor for Crohn disease, a major form of chronic inflammatory bowel disease. However, it is still unknown how the Atg16L1 variant contributes to disease development. The Atg16L1 protein possesses a C-terminal WD repeat domain whose function is entirely unknown, and the Crohn disease-associated mutation (T300A) is within this domain. To elucidate the function of the WD repeat domain, we established an experimental system in which a WD repeat domain mutant of Atg16L1 is stably expressed in Atg16L1-deficient mouse embryonic fibroblasts. Using the system, we show that the Atg16L1 complex forms a dimeric complex and that the total Atg16L1 protein level is strictly maintained, possibly by the ubiquitin proteasome system. Furthermore, we show that an Atg16L1 WD repeat domain deletion and the T300A mutant have little impact on canonical autophagy and autophagy against Salmonella enterica serovar Typhimurium. Therefore, we propose that Atg16L1 T300A is differentially involved in Crohn disease and canonical autophagy.     

Ku and DNA-dependent Protein Kinase Dynamic Conformations and Assembly Regulate DNA Binding and the Initial Non-homologous End Joining Complex

DNA double strand break (DSB) repair by non-homologous end joining (NHEJ) is initiated by DSB detection by Ku70/80 (Ku) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) recruitment, which promotes pathway progression through poorly defined mechanisms. Here, Ku and DNA-PKcs solution structures alone and in complex with DNA, defined by x-ray scattering, reveal major structural reorganizations that choreograph NHEJ initiation. The Ku80 C-terminal region forms a flexible arm that extends from the DNA-binding core to recruit and retain DNA-PKcs at DSBs. Furthermore, Ku- and DNA-promoted assembly of a DNA-PKcs dimer facilitates trans-autophosphorylation at the DSB. The resulting site-specific autophosphorylation induces a large conformational change that opens DNA-PKcs and promotes its release from DNA ends. These results show how protein and DNA interactions initiate large Ku and DNA-PKcs rearrangements to control DNA-PK biological functions as a macromolecular machine orchestrating assembly and disassembly of the initial NHEJ complex on DNA.     

p27Kip1 and Cyclin Dependent Kinase 2 Regulate Passage Through the Restriction Point

When quiescent cells are stimulated to re-enter the cell cycle, growth factors are required only until the restriction point in G1 phase. After this point the cell no longer requires growth factors, proliferative signaling molecules, or even protein synthesis in order to initiate DNA synthesis, which starts several hours later. Consequently, understanding the molecular nature of the restriction point constitutes one of the major goals in studies of growth regulation. We recently demonstrated that p27Kip1 (p27) regulates passage through G1 phase in actively proliferating cultures, and initiated these studies to determine if it is also involved in passage through the restriction point following stimulation of quiescent cells. In support of this suggestion, we found that passage through the restriction point requires mitogen-dependent suppression of the high p27 levels normally present in quiescent cells. Moreover, as the culture progresses to mid-G1 phase, the proportion of cells that pass the restriction point is increased by artificial suppression of p27 levels, while this proportion is reduced by elevation of p27 levels. p27 performs this critical function by regulating the subsequent activating phosphorylation of cyclin dependent kinase (CDK)2, which we also show is necessary for and closely associated with the initiation of DNA synthesis. We conclude that the p27 expression level at mid-G1 phase determines when a cell passes through the restriction point, and does so by regulating subsequent CDK2 activation.     

Phosphorylation of Rac1 T108 by Extracellular Signal-Regulated Kinase in Response to Epidermal Growth Factor: a Novel Mechanism To Regulate Rac1 Function

Accumulating evidence has implicated Rho GTPases, including Rac1, in many aspects of cancer development. Recent findings suggest that phosphorylation might further contribute to the tight regulation of Rho GTPases. Interestingly, sequence analysis of Rac1 shows that Rac1 T108 within the ¹⁰⁶PNTP¹⁰⁹ motif is likely an extracellular signal-regulated kinase (ERK) phosphorylation site and that Rac1 also has an ERK docking site, ¹⁸³KKRKRKCLLL¹⁹² (D site), at the C terminus. Indeed, we show here that both transfected and endogenous Rac1 interacts with ERK and that this interaction is mediated by its D site. Green fluorescent protein (GFP)-Rac1 is threonine (T) phosphorylated in response to epidermal growth factor (EGF), and EGF-induced Rac1 threonine phosphorylation is dependent on the activation of ERK. Moreover, mutant Rac1 with the mutation of T108 to alanine (A) is not threonine phosphorylated in response to EGF. In vitro ERK kinase assay further shows that pure active ERK phosphorylates purified Rac1 but not mutant Rac1 T108A. We also show that Rac1 T108 phosphorylation decreases Rac1 activity, partially due to inhibiting its interaction with phospholipase C-γ1 (PLC-γ1). T108 phosphorylation targets Rac1 to the nucleus, which isolates Rac1 from other guanine nucleotide exchange factors (GEFs) and hinders Rac1''s role in cell migration. We conclude that Rac1 T108 is phosphorylated by ERK in response to EGF, which plays an important role in regulating Rac1.