Dynein light chain 1 peptide inhibits human immunodeficiency virus infection in eukaryotic cells

Human immunodeficiency virus (HIV) uses kinases such as Pak1 and macropinocytosis for a productive infection. Recently dynein light chain 1 (DLC1), a component of the dynein motor, was identified as a Pak1 substrate and interacted with the C-terminal region of DLC1 (aa 61-89). The dynein motor is implicated in retrograde transport, also of HIV, to the nucleus. It is known that DLC1 is important in macropinocytosis, and anti-dynein antibodies inhibit a productive HIV infection. Here, we show that in Hela-β-gal cells macropinocytosis was effectively blocked by a peptide spanning the C-terminal 19 amino acids of DLC1. We also found that the DLC1 peptide was capable of inhibiting the early entry steps of HIV, and the DLC1 peptide efficiently inhibited a productive HIV infection, and cooperated with the anti-HIV activity of CD4 antibodies. Taken together, the potentially therapeutic DLC1 peptide represents an interesting class of HIV inhibitors, targeting an essential cellular component for HIV infection. Our findings raise the possibility that the use of a DLC1 peptide in combination with currently used anti-HIV agents, might offer additional arsenal against HIV infection in human cells.     

Stage-specific expression of dynein light chain-1 and its interacting kinase, p21-activated kinase-1, in rodent testes: implications in spermiogenesis.

Mammalian spermatogenesis is a complex process involving regulatory interactions of many gene products. In this study, we found that dynein light chain-1 (DLC1), a component of the dynein motor complex, is highly expressed in mouse and rat testes. Immunohistochemically detectable levels of DLC1 are observed specifically in spermatids in steps 9-16 in distinct subcellular compartments: in steps 9-11, DLC1 is predominantly localized in the nucleus; in steps 12 and 13, it is found in both nucleus and cytoplasm; and in step 14-16, it is present exclusively in the cytoplasm. In addition, we found p21-activated kinase 1 (Pak1), a protein kinase that activates DLC1 by phosphorylating DLC1 at Serine 88, was also expressed during these stages of spermatogenesis. Pak1 was also expressed in Leydig cells, in preleptotene primary spermatocytes, and in round spermatids. The spermiogenic stage-specific expression of DLC1 suggests a role for DLC1 in chromatin condensation, spermatid shaping, and the final release of sperm from the spermatogenic epithelium. Further, Pak1 may also play a role in spermiogenesis by regulating DLC1 phosphorylation and, consequently, its function.     

Serine 88 phosphorylation of the 8-kDa dynein light chain 1 is a molecular switch for its dimerization status and functions

Dynein light chain 1 (DLC1, also known as DYNLL1, LC8, and PIN), a ubiquitously expressed and highly conserved protein, participates in a variety of essential intracellular events. Transition of DLC1 between dimer and monomer forms might play a crucial role in its function. However, the molecular mechanism(s) that control the transition remain unknown. DLC1 phosphorylation on Ser(88) by p21-activated kinase 1 (Pak1), a signaling nodule, promotes mammalian cell survival by regulating its interaction with Bim and the stability of Bim. Here we discovered that phosphorylation of Ser(88), which juxtapose each other at the interface of the DLC dimer, disrupts DLC1 dimer formation and consequently impairs its interaction with Bim. Overexpression of a Ser(88) phosphorylation-inactive DLC1 mutant in mammary epithelium cells and in a transgenic animal model caused apoptosis and accelerated mammary gland involution, respectively, with increased Bim levels. Structural and biophysical studies suggested that phosphorylation-mimicking mutation leads to dissociation of the DLC1 dimer to a pure folded monomer. The phosphorylation-induced DLC1 monomer is incapable of binding to its substrate Bim. These findings reveal a previously unrecognized regulatory mechanism of DLC1 in which the Ser(88) phosphorylation acts as a molecular switch for the transition of DLC1 from dimer to monomer, thereby modulating its interaction with substrates and consequently regulating the functions of DLC1.     

p21-activated Kinase 1 (Pak1)-dependent phosphorylation of Raf-1 regulates its mitochondrial localization, phosphorylation of BAD, and Bcl-2 association

Raf-1 protects cells from apoptosis, independently of its signals to MEK and ERK, by translocating to the mitochondria where it binds Bcl-2 and displaces BAD. However, the answer to the question of how Raf-1 is normally lured to the mitochondria and becomes activated remains elusive. p21-activated protein kinases (Paks) are serine/threonine protein kinases that phosphorylate Raf-1 at Ser-338 and Ser-339. Here we elucidate the molecular mechanism through which Pak1 signals to BAD through a Raf-1-activated pathway. Upon phosphorylation by Pak1, Raf-1 translocates to mitochondria and phosphorylates BAD at Ser-112. Moreover, the mitochondrial translocation of Raf-1 and the interaction between Raf-1 and Bcl-2 are regulated by Raf-1 phosphorylation at Ser-338/Ser-339. Notably, we show that formation of a Raf-1-Bcl-2 complex coincides with loss of an interaction between Bcl-2 and BAD. These signals are specific for Pak1, because Src-activated Raf-1 only stimulates the MAP kinase cascade. Thus, our data identify the molecular connections of a Pak1-Raf-1-BAD pathway that is involved in cell survival signaling.     

Regulation of the interaction of Pak2 with Cdc42 via autophosphorylation of serine 141

Pak2, a member of the p21-activated protein kinase (Pak) family, is activated in response to a variety of stresses and is directly involved in the induction of cytostasis. At the molecular level Pak2 binds Cdc42(GTP), translocating Pak2 to the endoplasmic reticulum where it is autophosphorylated and activated. Pak2 is autophosphorylated at eight sites; Ser-141 and Ser-165 in the regulatory domain and Thr-402 in the activation loop are identified as key sites in activation of the protein kinase. The function of phosphorylation of Ser-141 and Ser-165 on the activation was analyzed with wild-type (WT) and mutants of Pak2. With S141A, the level of autophosphorylation was reduced to 65% as compared with that of WT and S141D with a concomitant 45% reduction in substrate phosphorylation, indicating that phosphorylation at Ser-141 is required for optimal activity. Autophosphorylation inhibited the interaction between WT Pak2 and Cdc42(GTP). In 293T cells, WT Pak2, S141A, and S141D formed a stable complex with the constitutively active mutant Cdc42 L61, but not with the dominant negative Cdc42 N17. As shown in glutathione S-transferase pull-down assays, S141A bound to Cdc42(GTP) at a 6-fold higher level than that of S141D. In contrast, the S165A and S165D mutants had no effect on autophosphorylation, binding to Cdc42, or activation of Pak2. In summary, autophosphorylation of Ser-141 was required for activation of Pak2 and down-regulated the interaction of Pak2 with Cdc42. A model is proposed suggesting that binding of Cdc42 localizes Pak2 to the endoplasmic reticulum, where autophosphorylation alters association of the two proteins.     

Biochemical and structural characterization of the Pak1-LC8 interaction

Pak1 (p21-activated kinase-1) and the dynein light chain, LC8, are overexpressed in breast cancer, and their direct interaction has been proposed to regulate tumor cell survival. These effects have been attributed in part to Pak1-mediated phosphorylation of LC8 at serine 88. However, LC8 is homodimeric, which renders Ser(88) inaccessible. Moreover, Pak1 does not contain a canonical LC8 binding sequence compared with other characterized LC8 binding sequences. Together, these observations raise the question whether the Pak1/LC8 interaction is distinct (i.e. enabled by a unique interface independent of LC8 dimerization). Herein, we present results from biochemical, NMR, and crystallographic studies that show that Pak1 (residues 212-222) binds to LC8 along the same groove as canonical LC8 interaction partners (e.g. nNOS and BimL). Using LC8 point mutants K36P and T67A, we were able to differentiate Pak1 from canonical LC8 binding sequences and identify a key hydrogen bond network that compensates for the loss of the conserved glutamine in the consensus sequence. We also show that the target binding interface formed through LC8 dimerization is required to bind to Pak1 and precludes phosphorylation of LC8 at Ser(88). Consistent with this observation, in vitro phosphorylation assays using activated Pak1 fail to phosphorylate LC8. Although these results define structural details of the Pak1/LC8 interaction and suggest a hierarchy of target binding affinities, they do not support the current model whereby Pak1 binds to and subsequently phosphorylates LC8 to promote anchorage-independent growth. Rather, they suggest that LC8 binding modulates Pak1 activity and/or nuclear localization.     

Membrane transport of WAVE2 and lamellipodia formation require Pak1 that mediates phosphorylation and recruitment of stathmin/Op18 to Pak1–WAVE2–kinesin complex

Membrane transport of WAVE2 that leads to lamellipodia formation requires a small GTPase Rac1, the motor protein kinesin, and microtubules. Here we explore the possibility of whether the Rac1-dependent and kinesin-mediated WAVE2 transport along microtubules is regulated by a p21-activated kinase Pak as a downstream effector of Rac1. We find that Pak1 constitutively binds to WAVE2 and is transported with WAVE2 to the leading edge by stimulation with hepatocyte growth factor (HGF). Concomitantly, phosphorylation of tubulin-bound stathmin/Op18 at serine 25 (Ser25) and Ser38, microtubule growth, and stathmin/Op18 binding to kinesin–WAVE2 complex were induced. The HGF-induced WAVE2 transport, lamellipodia formation, stathmin/Op18 phosphorylation at Ser38 and binding to kinesin–WAVE2 complex, but not stathmin/Op18 phosphorylation at Ser25 and microtubule growth, were abrogated by Pak1 inhibitor IPA-3 and Pak1 depletion with small interfering RNA (siRNA). Moreover, stathmin/Op18 depletion with siRNA caused significant inhibition of HGF-induced WAVE2 transport and lamellipodia formation, with HGF-independent promotion of microtubule growth. Collectively, it is suggested that Pak1 plays a critical role in HGF-induced WAVE2 transport and lamellipodia formation by directing Pak1–WAVE2–kinesin complex toward the ends of growing microtubules through phosphorylation and recruitment of tubulin-bound stathmin/Op18 to the complex.     

cGMP-dependent protein kinase phosphorylates p21-activated kinase (Pak) 1, inhibiting Pak/Nck binding and stimulating Pak/vasodilator-stimulated phosphoprotein association

Endothelial cells are normally non-motile and quiescent; however, endothelial cells will become permeable and invade and proliferate to form new blood vessels (angiogenesis) in response to wounding, cancer, diabetic retinopathy, age-related macular degeneration, or rheumatoid arthritis. p21-activated kinase (Pak), an effector for the Rho GTPases Rac and Cdc42, is required for angiogenesis and regulates endothelial cell permeability and motility. Although Pak is primarily activated by Rac and Cdc42, there are additional proteins that regulate Pak activity and localization, including three AGC protein kinase family members, Akt-1, PDK-1, and cAMP-dependent protein kinase. We describe phosphorylation and regulation of Pak localization by a fourth AGC kinase family member, cGMP-dependent protein kinase (PKG). Using in vitro mapping, a phosphospecific antibody, co-transfection assays, and untransfected bovine aortic endothelial cells we determined that PKG phosphorylates Pak at serine 21. Phosphorylation was accompanied by changes in proteins associated with Pak. The adaptor protein Nck was released, whereas a novel complex with vasodilator-stimulated phosphoprotein was stimulated. Furthermore Ser-21 phosphorylation of Pak appears to be important for regulation of cell morphology. In both human umbilical vein endothelial cells and HeLa cells, activation of PKG in the presence of Pak stimulated tail retraction and cell polarization. However, in cells expressing S21A mutant Pak1, PKG activation or treatment with a peptide that blocks Nck/Pak binding caused aberrant cell morphology, blocked cell retraction, and mislocalized Pak, producing uropod (tail-like) structures. These data suggest that PKG regulates Pak and that the interaction plays a role in tail retraction.     

Motor Domain Phosphorylation Modulates Kinesin-1 Transport

Disruptions in microtubule motor transport are associated with a variety of neurodegenerative diseases. Post-translational modification of the cargo-binding domain of the light and heavy chains of kinesin has been shown to regulate transport, but less is known about how modifications of the motor domain affect transport. Here we report on the effects of phosphorylation of a mammalian kinesin motor domain by the kinase JNK3 at a conserved serine residue (Ser-175 in the B isoform and Ser-176 in the A and C isoforms). Phosphorylation of this residue has been implicated in Huntington disease, but the mechanism by which Ser-175 phosphorylation affects transport is unclear. The ATPase, microtubule-binding affinity, and processivity are unchanged between a phosphomimetic S175D and a nonphosphorylatable S175A construct. However, we find that application of force differentiates between the two. Placement of negative charge at Ser-175, through phosphorylation or mutation, leads to a lower stall force and decreased velocity under a load of 1 piconewton or greater. Sedimentation velocity experiments also show that addition of a negative charge at Ser-175 favors the autoinhibited conformation of kinesin. These observations imply that when cargo is transported by both dynein and phosphorylated kinesin, a common occurrence in the cell, there may be a bias that favors motion toward the minus-end of microtubules. Such bias could be used to tune transport in healthy cells when properly regulated but contribute to a disease state when misregulated.     

The 8-kDa dynein light chain binds to its targets via a conserved (K/R)XTQT motif

Cytoplasmic dynein is a large, multisubunit molecular motor that translocates cargoes toward the minus ends of microtubules. Proper functioning of the dynein motor requires precise assembly of its various subunits. Using purified recombinant proteins, we show that the highly conserved 8-kDa light chain (DLC8) binds to the intermediate chain of the dynein complex. The DLC8-binding region was mapped to a highly conserved 10-residue fragment (amino acid sequence SYSKETQTPL) C-terminal to the second alternative splicing site of dynein intermediate chain. Yeast two-hybrid screening using DLC8 as bait identified numerous additional DLC8-binding proteins. Biochemical and mutational analysis of selected DLC8-binding proteins revealed that DLC8 binds to a consensus sequence containing a (K/R)XTQT motif. The (K/R)XTQT motif interacts with the common target-accepting grooves of DLC8 dimer. The role of each conserved amino acid residue in this pentapeptide motif in supporting complex formation with DLC8 was systematically studied using site-directed mutagenesis.     

NMR characterization of structural and dynamics perturbations due to a single point mutation in Drosophila DLC8 dimer: functional implications

Dynein light chain protein (DLC8), the smallest subunit of the dynein motor complex, acts as a cargo adaptor. The protein exists as a dimer under physiological conditions, and cargo binding occurs at the dimer interface. Dimer stability and relay of perturbations through the dimer interface can thus be anticipated to play crucial roles in the variety of functions the protein performs. Recent investigations point out that DLC8 also gets phosphorylated at Ser 88, which is located at the extreme C-terminal end. In this background, we investigate here by NMR the effects of a small perturbation by way of a single point mutation, S88A, on the structure, dynamics, and cargo binding efficacy of the DLC8 dimer. We observe that the perturbation travels far away along the sequence from the site of the mutation. This relay has been explained at the atomic level by looking into the packing of the side chains in the crystal structure of the protein. It follows that the interface is highly adaptable, which may account for the versatility of the dimer's cargo binding ability. Binding studies with a peptide indicate that the mutation compromises binding efficacy. These observations show how remote residues that may not be directly bound to a target can still affect the affinity of the protein to the target. Furthermore, the S88A mutational perturbations seen here in Drosophila DLC8 are dramatically different from those of the same mutation in human DLC8 (also known as DLC1) ( Song, C. , et al., ( 2008) J. Biol. Chem, 283, 4004- 4013. ) which differs from Drosophila DLC8 at only five locations. All of these observations put together highlight the sensitivity of dynein light chain protein to small perturbations, and this would have great functional implications.     

Unleashing the Ambra1-Beclin 1 complex from dynein chains: Ulk1 sets Ambra1 free to induce autophagy

The Beclin 1-VPS34 complex plays a crucial role in the induction of the autophagic process by generating PtdIns(3)P-rich membranes, which act as platforms for ATG protein recruitment and autophagosome nucleation. Several cofactors, such as Ambra1, ATG14 and UVRAG, are necessary for Beclin 1 complex activity. However, the mechanism by which Beclin 1 complex activity is: stimulated by autophagic stimuli has not yet been fully elucidated. Recently, we reported that autophagosome formation in mammalian cells is primed by Ambra1 release from the dynein motor complex. We found that Ambra1 specifically binds the dynein motor complex under normal conditions through a direct interaction with DLC1. When autophagy is induced, Ambra1-DLC1 are released from the dynein complex in an ULK1-dependent manner, and relocalize to the endoplasmic reticulum, thus enabling autophagosome nucleation. In addition, we found that both DLC1 downregulation and Ambra1 mutations in its DLC1-binding sites strongly enhance autophagosome formation. Ambra1 is therefore not only a cofactor of Beclin 1 in favoring its kinase-associated activity, but also a crucial upstream regulator of autophagy initiation.     

p21-Activated kinase 5 (Pak5) localizes to mitochondria and inhibits apoptosis by phosphorylating BAD

Pak5 is the most recently identified and least understood member of the p21-activated kinase (Pak) family. This kinase is known to promote neurite outgrowth in vitro, but its localization, substrates, and effects on cell survival have not been reported. We show here that Pak5 has unique properties that distinguish it from all other members of the Pak family. First, Pak5, unlike Pak1, cannot complement an STE20 mutation in Saccharomyces cerevisiae. Second, Pak5 binds to the GTPases Cdc42 and Rac, but these GTPases do not regulate Pak5 kinase activity, which is constitutive and stronger than any other Pak. Third, Pak5 prevents apoptosis induced by camptothecin and C2-ceramide by phosphorylating BAD on Ser-112 in a protein kinase A-independent manner and prevents the localization of BAD to mitochondria, thereby inhibiting the apoptotic cascade that leads to apoptosis. Finally, we show that Pak5 itself is constitutively localized to mitochondria, and that this localization is independent of kinase activity or Cdc42 binding. These features make Pak5 unique among the Pak family and suggest that it plays an important role in apoptosis through BAD phosphorylation.     

Filamin is essential in actin cytoskeletal assembly mediated by p21-activated kinase 1

The serine/threonine kinase p21-activated kinase 1 (Pak1) controls the actin cytoskeletal and ruffle formation through mechanisms that are independent of GTPase activity. Here we identify filamin FLNa as a Pak1-interacting protein through a yeast two-hybrid screen using the amino terminus of Pak1 as a bait. FLNa is stimulated by physiological signalling molecules to undergo phosphorylation by Pak1 and to interact and colocalize with endogenous Pak1 in membrane ruffles. The ruffle-forming activity of Pak1 is functional in FLNa-expressing cells but not in FLNa-deficient cells. In FLNa, the Pak1-binding site involves tandem repeat 23 in the carboxyl terminus and phosphorylation takes place on serine 2152. The FLNa-binding site in Pak1 is localized between amino acids 52 and 132 in the conserved Cdc42/Rac-interacting (CRIB) domain; accordingly, FLNa binding to the CRIB domain stimulates Pak1 kinase activity. Our results indicate that FLNa may be essential for Pak1-induced cytoskeletal reorganization and that the two-way regulatory interaction between Pak1 and FLNa may contribute to the local stimulation of Pak1 activity and its targets in cytoskeletal structures.     

Cell cycle-regulated phosphorylation of p21-activated kinase 1

Mammalian p21-activated kinase 1 (Pak1) is a highly conserved effector for the small GTPases Cdc42 and Rac1. In lower eukaryotes, Pak1 homologs are regulated during the cell cycle by phosphorylation. Here, we show that Pak1 is phosphorylated during mitosis in mammalian fibroblasts. This phosphorylation occurs at a single site, Thr 212, within a domain that is unique to Pak1. Cdc2 phosphorylates Pak1 at the identical site in vitro, and inhibition of Cdc2 abolishes Pak1 mitotic phosphorylation in vivo, indicating that Cdc2 is the kinase responsible for phosphorylating Pak1 in mitotic cells. Expression of a Pak1 mutant in which Thr 212 is replaced with a phosphomimic (aspartic acid) has marked effects on the rate and extent of postmitotic spreading of fibroblasts. The mitotic phosphorylation of Pak1 does not alter the basal or Rac-stimulated activity of this kinase, but it does affect the coimmunoprecipitation of at least three proteins with Pak1. These findings are the first to implicate a mammalian Pak in cell cycle regulation and suggest that Pakl, as a result of phosphorylation by Cdc2, alters its association with binding partners and/or substrates that are relevant to the morphologic changes associated with cell division.     

Identification of p122RhoGAP (deleted in liver cancer-1) Serine 322 as a substrate for protein kinase B and ribosomal S6 kinase in insulin-stimulated cells

Protein kinase B (PKB or Akt) plays an essential role in the actions of insulin, cytokines, and growth factors, although the substrates for PKB that are relevant to many of its actions require identification. In this study, we have reported the identification of p122RhoGAP, a GTPase-activating protein selective for RhoA and rodent homologue of the tumor suppressor deleted in liver cancer (DLC1) as a novel insulin-stimulated phosphoprotein in primary rat adipocytes. We have demonstrated that Ser-322 is phosphorylated upon insulin stimulation of intact cells and that this site is directly phosphorylated in vitro by PKB and ribosomal S6 kinase, members of the AGC (protein kinases A, G, and C) family of insulin-stimulated protein kinases. Furthermore, expression of constitutively active mutants of PKB or mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) stimulates Ser-322 phosphorylation in intact cells, demonstrating that activation of the PKB or MEK pathway is sufficient for Ser-322 phosphorylation in vivo. Indeed, in primary adipocytes, insulin-stimulated Ser-322 phosphorylation was almost exclusively regulated by the phosphatidylinositol 3-kinase/PKB pathway, whereas in immortalized cells, insulin-stimulated phosphorylation was predominantly regulated by the MEK/extracellular signal-regulated kinase/ribosomal S6 kinase pathway, with the phosphatidylinositol 3-kinase/PKB pathway playing a minor role. These results demonstrate that p122RhoGAP Ser-322 acts as an integrator of signal transduction in a manner dependent on the cellular context.     

Mitotic phosphorylation of the dynein light intermediate chain is mediated by cdc2 kinase

Cytoplasmic dynein, a large minus-end-directed microtubule motor, performs multiple functions during the cell cycle. In interphase, dynein moves membrane organelles, while in mitosis it moves chromosomes and helps to form the mitotic spindle. The cell-cycle regulation of dynein activity may be controlled, at least in part, by the phosphorylation of its light intermediate chains (DLIC), since a 10-fold increase in light intermediate chain phosphorylation correlates with a decrease in dynein-based membrane transport of similar magnitude in mitosis. In this study, we sought to identify the kinase responsible for this potentially important phosphorylation event. We show that bacterially-expressed chicken light intermediate chain (chDLIC) will undergo mitosis-specific phosphorylation when added to Xenopus egg extracts. Mutation of a conserved cdc2 kinase consensus site (Ser197) abolishes this phosphorylation event, and mass spectroscopy analysis confirms that the wild-type DLIC is stoichiometrically phosphorylated at this site when incubated with metaphase but not interphase extracts. We also show that purified cdc2 kinase phosphorylates purified DLICs at Ser197 in vitro and that Ser197 phosphorylation is dramatically reduced in metaphase extracts depleted of cdc2 kinase. These results indicate that cdc2 kinase directly phosphorylates dynein and thus may be an important regulator of dynein activity in the cell cycle.     

p21-Activated Kinase 1 (PAK1) Can Promote ERK Activation in a Kinase-independent Manner

PAK1 plays an important role in proliferation and tumorigenesis, at least partially by promoting ERK phosphorylation of C-RAF (Ser-338) or MEK1 (Ser-298). We observed how that overexpression of a kinase-dead mutant form of PAK1 increased phosphorylation of MEK1/2 (Ser-217/Ser-221) and ERK (Thr-202/Tyr-204), although phosphorylation of B-RAF (Ser-445) and C-RAF (Ser-338) remained unchanged. Furthermore, increased activation of the PAK1 activator Rac1 induced the formation of a triple complex of Rac1, PAK1, and MEK1 independent of the kinase activity of PAK1. These data suggest that PAK1 can stimulate MEK activity in a kinase-independent manner, probably by serving as a scaffold to facilitate interaction of C-RAF.     

Phosphorylation of amphiphysin I by minibrain kinase/dual-specificity tyrosine phosphorylation-regulated kinase, a kinase implicated in Down syndrome

Minibrain kinase/dual-specificity tyrosine phosphorylation-regulated kinase (Mnb/Dyrk1A) is a proline-directed serine/threonine kinase encoded in the Down syndrome critical region of human chromosome 21. This kinase has been shown to phosphorylate dynamin 1 and synaptojanin 1. Here we report that amphiphysin I (Amph I) is also a Mnb/Dyrk1A substrate. This kinase phosphorylated native Amph I in rodent brains and recombinant human Amph I expressed in Escherichia coli. Serine 293 (Ser-293) was identified as the major site, whereas serine 295 and threonine 310 were found as minor kinase sites. In cultured cells, recombinant Amph I was phosphorylated at Ser-293 by endogenous kinase(s). Because mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) has been suggested to phosphorylate Amph I at Ser-293, our efforts addressed whether Ser-293 is phosphorylated in vivo by MAPK/ERK or by Mnb/Dyrk1A. Overnight serum-withdrawal inactivated MAPK/ERK; nonetheless, Ser-293 was phosphorylated in Chinese hamster ovary and SY5Y cells. Epigallocatechin-3-gallate, a potent Mnb/Dyrk1A inhibitor in vitro, apparently reduced the phosphorylation at Ser-293, whereas PD98059, a potent MAPK/ERK inhibitor, did not. High frequency stimulation of mouse hippocampal slices reduced the phosphorylation at Ser-293, albeit in the midst of MAPK/ERK activation. The endophilin binding in vitro was inhibited by phosphorylating Amph I with Mnb/Dyrk1A. However, phosphorylation at Ser-293 did not appear to alter cellular distribution patterns of the protein. Our results suggest that Mnb/Dyrk1A, not MAPK/ERK, is responsible for in vivo phosphorylation of Amph I at Ser-293 and that phosphorylation changes the recruitment of endophilin at the endocytic sites.