THE MECHANISM OF ACTION OF SPROUTY2: CHARACTERIZATION OF THE INTERACTION BETWEEN SPROUTY2 AND PKCδ CHOW SOAH YEE B.Sc. (Hons) National University of Singapore A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY INSTITUTE OF MOLECULAR AND CELL BIOLOGY DEPARTMENT OF PHYSIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2009 ACKNOWLEDGEMENTS I would like to express my sincerest gratitude to my supervisor A/Prof Graeme Guy for his advice, guidance and understanding throughout my PhD candidature. My thanks also extend to my PhD supervisory committee members, A/Prof Cao Xinmin and A/Prof Low Boon Chuan, for their suggestions and advice. My heartfelt gratitude to Dr Permeen Yusoff, Dr Sumana Chandramouli and Dr Lao Dieu Hung for their help, guidance, advice, discussions and friendship. Special thanks also to Yu Chye Yun for her tireless assistance. I would like to thank all the members of the GG lab, past and present, for having created a friendly and interesting environment to work in, especially Dr Ben McCaw, for his training and patience early in my candidature and Judy Saw for her advice on immunofluoroscence. I would also like to thank Dr Permeen Yusoff and Dr Rebecca Jackson for their patient proofreading of this thesis, despite their busy schedules. On a personal note, I would like to thank my family and friends for putting up with me and encouraging me the last few years. Special thanks go to Dr Louis Payet, for his constant support and encouragement. I would like to express my appreciation to Hanping, Ellen and Yen Li for their concern. I am also grateful to my nephew and niece, Calvin and Casie, for providing everyday comic relief. Finally, I would like to dedicate this thesis to those who have always believed in me. Chow Soah Yee June 2009 I TABLE OF CONTENTS ACKNOWLEDGEMENTS I TABLE OF CONTENTS II LIST OF FIGURES VIII LIST OF TABLES X ABBREVIATIONS XI SUMMARY XIV Chapter Introduction 1.1 Cell signaling 1.2 Receptor tyrosine kinase signaling 1.2.1 Activation of RTKs 1.2.2 Intracellular pathways Overview of the Ras-ERK signaling pathway 1.3.1 Components of the Ras-ERK signaling pathway 1.3.1.1 Grb2 1.3.1.2 Sos 1.3.1.3 Ras 10 1.3.1.4 Raf 13 1.3.1.5 MEK 17 1.3.1.6 ERK 18 1.3 1.3.1.6.1 ERK substrates 18 1.3.1.6.2 Scaffolds 19 1.4 Regulation of Ras-ERK signaling 20 1.5 The ERK pathway in cancer 23 1.6 Sprouty 24 II 1.6.1 Sprouty as an inhibitor of RTK signaling 24 1.6.2 Primary structure of Spry 25 1.6.3 Post-translational modifications on Spry 26 1.6.4 Sprouty localization 28 1.6.5 Inhibition of the Ras-ERK signaling pathway by Spry 29 1.6.6 Points of action of Spry 30 1.6.6.1 Upstream of Ras 30 1.6.6.2 Upstream of Raf 32 1.6.7 Regulation of receptor ubiquitination and endocytosis by Spry2 32 1.6.8 Physiological functions 34 1.6.8.1 Inhibition of cell proliferation and migration 34 1.6.8.2 Inhibition of branching morphogenesis 35 1.6.8.3 Role of Sprouty in development 36 Sprouty in cancer 37 1.6.9 1.7 1.8 1.9 PLCγ-PKC signaling 38 1.7.1 38 Primary structure of PLCγ 1.7.2 Activation of PLCγ 40 The protein kinase C family 42 1.8.1 Primary structure of PKC 42 1.8.2 Phosphorylation on PKCδ 43 1.8.3 Activation of PKCδ 45 1.8.4 Scaffolds 47 1.8.5 47 Regulation of PKC signaling 1.8.6 Substrates of PKCδ and downstream signaling 48 1.8.7 49 Physiological functions of PKCδ 1.8.8 PKCδ in disease 50 Protein Kinase D (PKD) 51 1.9.1 51 Primary structure of PKD 1.9.2 Activation and phosphorylation of PKD1 52 1.9.3 Substrates and downstream signaling 54 1.9.4 Physiological functions of PKD1 55 III 1.10 Research Objectives Chapter Materials and Methods 56 58 2.1 Chemicals and Reagents 58 2.2 Antibodies 58 2.3 Plasmid constructs 59 2.3.1 59 2.4 2.5 2.6 Phospholipid Scramblase 2.3.2 PKCδ 59 2.3.3 PKD1 59 2.3.4 Other plasmid constructs 60 DNA methodology 60 2.4.1 Expression vectors 60 2.4.2 Agarose gel electrophoresis 61 2.4.3 Restriction enzyme digestion and ligation 61 2.4.4 Electrocompetent E. coli cell preparation 62 2.4.5 Electroporation 62 2.4.6 Plasmid DNA preparation 63 2.4.7 Polymerase Chain Reaction (PCR) 63 2.4.8 DNA purification 64 2.4.9 64 Reverse Transcriptase PCR 2.4.10 Site-directed mutagenesis 65 Cell culture 66 2.5.1 Maintenance and propagation of mammalian cells 66 2.5.2 Mammalian cell transfection 66 2.5.3 Serum starvation and growth factor stimulation of mammalian cells 67 2.5.4 UV irradiation 67 Protein biochemistry 68 2.6.1 68 Preparation of cell extracts 2.6.2 Protein quantification 68 2.6.3 Immunoprecipitation 69 IV 2.6.4 SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE) 69 2.6.5 Western blot (immunoblot) analysis 70 2.6.6 Expression and purification of GST-fusion proteins 71 2.6.7 Far-Western assay 72 2.6.8 Small hairpin RNA knockdown 73 2.7 Immunofluorescence 73 2.8 Cell invasion assay 74 2.9 Yeast two-hybrid screen 75 2.9.1 Cloning and transformation of bait 76 2.9.2 Verification of protein expression 77 2.9.3 Interaction control 77 2.9.4 Toxicity testing 78 2.9.5 Self-activation test 78 2.9.6 Yeast library screening 79 2.9.7 Large scale plasmid isolation 79 2.9.8 Sequencing and identification of isolated clones 80 2.9.9 Subcloning of in-frame clones 81 2.9.10 Expression and binding in mammalian cells Chapter 3.1 3.2 Results 81 82 Characterizing the interaction between Spry2 and its interacting partners 82 3.1.1 Yeast two-hybrid screen to identify interacting partners of Spry2 82 3.1.2 Cloning of Plscr3 and its interaction with Spry proteins 83 3.1.3 Spry2 interacts with Plscr3 mainly through its C-terminal domain 87 3.1.4 Plscr3 interacts with PKCδ upon UV irradiation 89 3.1.5 Discussion and Conclusions 89 Characterization of interaction between Spry2 and PKCδ 91 3.2.1 Spry2 interacts with PKCδ 91 3.2.2 Spry2 interacts specifically with PKCδ 93 3.2.3 Interaction between Spry2 and PKCδ occurs with acute FGF stimulation 93 V 3.3 3.4 3.5 3.2.4 PKCδ and Spry2 form a complex endogenously 95 3.2.5 Full-length Spry2 is required for its interaction with PKCδ 95 3.2.6 Interaction between Spry2 and PKCδ is conformation dependent 98 3.2.7 106 Spry2 co-localizes with PKCδ Mechanism of action of Spry2 110 3.3.1 Spry2 interacts with PLCγ1 110 3.3.2 Spry2 does not affect phosphorylation of T505 on PKCδ 114 3.3.3 Spry2 prevents the phosphorylation of PKD1 by PKCδ 114 3.3.4 Spry2 interacts more strongly with PKCδ in the presence of PKD1 118 3.3.5 Spry2 co-localizes with both PKCδ and PKD1 123 3.3.6 Spry2 binds directly to both PKCδ and PKD1 127 3.3.7 Spry2 requires the ATP-binding site on PKCδ for the interaction 127 3.3.8 Spry2 requires a PKCδ-PKD1 interaction to associate with PKCδ 131 3.3.9 Summation 133 Effect of Spry2 on PKCδ signaling in ERK1/2 activation 135 3.4.1 PKCδ contributes to ERK1/2 activity 135 3.4.2 Kinase activity of PKCδ is required for ERK1/2 phosphorylation 137 3.4.3 Spry is able to inhibit ERK1/2 phosphorylation downstream of PKCδ 137 3.4.4 Spry2 increases RIN1 interaction with Ras 139 Effect of Spry2 on PKCδ signaling in cell invasion 143 3.5.1 PKCδ affects cell invasion of PC-3 cells 143 3.5.2 145 Spry2 reduces invasion of PC-3 cells 3.5.3 PKCδ is able to reverse the effect of Spry on PC-3 cell invasion Chapter Discussion 145 149 4.1 Conformation as a factor in the interaction specificity of Spry2 and PKCδ 150 4.2 Spry2 doesnot inhibit the phosphorylation of PKCδ by an upstream kinase 150 4.3 Implications of PLCγ1-Spry interaction 151 4.4 Formation of a Spry2-PKCδ-PKD1 complex: is Spry2 a PKCδ substrate? 152 VI 4.5 The function of the PKCδ C2 domain in the PKCδ-PKD1 interaction 153 4.6 The impact of Spry2 on HDAC5 154 4.7 Spry2 inhibits ERK1/2 phosphorylation via PKCδ and PKD1 155 4.8 The implications of Spry2 on signaling downstream of RIN1 157 4.8.1 RIN1 and Abl 157 4.8.2 RIN1 and Rab5 157 4.9 A working model for the Spry2-PKCδ-PKD1 interaction 158 4.10 Areas for future research 161 Addendum 163 References 164 Publications 185 VII LIST OF FIGURES 1.2 Domain organization of different RTK classes 1.3.1 Schematic representation of the Ras-ERK pathway induced by RTKs 1.3.1.3 The primary structure of Ras isoforms 12 1.3.1.4 Schematic representation of the primary structure of Raf 15 1.6.2 The structure and isoforms of Spry 27 1.7 Schematic representation of PLCγ signaling 39 1.7.1 Schematic representation of the primary structure of PLCγ1 41 1.8.1 Primary structure of PKCδ 44 1.9.1 Primary structure of PKD1 53 2.1 Multiple cloning site sequence of pXJ40 epitope tagged vector 60 2.2 Summary of the cycling program used for PCR 64 3.1.1 Representative results of binding partners to Sprouty2 isolated in the yeast 85 two-hybrid screen 3.1.2 Plscr3 interacts with all the Spry isoforms 86 3.1.3 Spry2 interacts with Plscr3 mainly through its C-terminal domain 88 3.1.4 Verification of PKCδ as an interaction partner of Plscr3 90 3.2.1 Sprouty2 interacts with PKCδ upon FGFR1 activation 92 3.2.2 Spry2 interacts specifically with PKCδ 94 3.2.3 Spry2 interacts with PKCδ upon FGF stimulation 96 3.2.4 Spry2 and PKCδ interact with each other endogenously 97 3.2.5 Construction of Spry2 truncated proteins 99 3.2.6 Full length Spry2 protein is required for interaction with PKCδ 100 3.2.7 Verification of the requirement of a full-length Spry2 protein for its interaction 101 with PKCδ 3.2.8 Full-length Spry2 interacts with both full-length and truncated PKCδ proteins 103 3.2.9 104 N-terminal Spry2 interacts with C-terminal PKCδ protein: the interaction is VIII conformation-dependent 3.2.10 C-terminal Spry2 interacts with both N- and C-terminal PKCδ: the interaction 105 is conformation-dependent 3.2.11 Determining the localization of Spry2 and PKCδ 107 3.2.12 Verification of the co-localization of Spry2 and PKCδ proteins 109 3.3.1 Spry2 interacts with PLCγ1 in a stimulation-dependent manner 111 3.3.2 Spry2 interacts with PLCγ1 through its N-terminal domain 113 3.3.3 Spry2 does not inhibit the phosphorylation of PKCδ by its upstream kinase 115 3.3.4 PKD1 is a substrate of PKCδ 117 3.3.5 Spry2 blocks PKCδ from phosphorylating its substrate PKD1 119 3.3.6 The two alternatives for the mechanism of action by Spry2 121 3.3.7 Increasing levels of PKD1 enhances the interaction between PKCδ and Spry2 122 3.3.8 Verification that PKD1 increases the interaction between Spry2 and PKCδ 124 3.3.9 125 Determining the localization of PKD1 in conjunction with PKCδ and Spry2 3.3.10 Verification of co-localization of PKD1, PKCδ and Spry2 126 3.3.11 Spry2 interacts directly with both PKCδ and PKD1 128 3.3.12 PKCδ-KD prevents the interaction between Spry2 and endogenous PKCδ 130 3.3.13 Increasing PKCδ-KD levels decrease the endogenous PKCδ-Spry2 interaction 132 3.3.14 Interaction between PKCδ and PKD1 is required for Spry2-PKCδ binding 134 3.4.1 PKCδ 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Atomic structure of the MAP kinase ERK2 at 2.3 Å resolution. Nature 367, 704-711. Zheng, C.F., and Guan, K.L. (1993). Properties of MEKs, the kinases that phosphorylate and activate the extracellular signal-regulated kinases. J Biol Chem 268, 23933-23939. Zheng, C.F., and Guan, K.L. (1994). Activation of MEK family kinases requires phosphorylation of two conserved Ser/Thr residues. EMBO J 13, 1123-1131. Zhou, G., Bao, Z.Q., and Dixon, J.E. (1995). Components of a new human protein kinase signal transduction pathway. J Biol Chem 270, 12665-12669. 184 PUBLICATIONS Soah Yee Chow, Chye Yun Yu, and Graeme R. Guy. Sprouty2 interacts with Protein Kinase Cδ and disrupts phosphorylation of Protein Kinase D 1. J Biol Chem 2009 May 19 (Epub ahead of print) Graeme R. Guy, Rebecca A. Jackson, Permeen Yusoff, and Soah Yee Chow. Sprouty proteins: modified modulators, matchmakers or missing links? J Endocrinol 2009 May 12 (Epub ahead of print) Benjamin J. McCaw*, Soah Yee Chow*, Esther S.M. Wong, Kwee Leng Tan, Huili Guo, and Graeme R. Guy (2005). Identification and characterization of mERK5-T, a novel Erk5/BMK1 splice variant. Gene 345, 183-190. *These authors contributed equally to the work. 185 Gene 345 (2005) 183 – 190 www.elsevier.com/locate/gene Identification and characterization of mErk5-T, a novel Erk5/Bmk1 splice variantB B.J. McCaw1, S.Y. Chow1, E.S.M. Wong, K.L. Tan, H. Guo, G.R. Guy* Signal Transduction Laboratory, Institute of Molecular and Cell Biology, Proteos, 61 Biopolis Drive, Singapore 138673, Singapore Received 24 September 2004; received in revised form 25 October 2004; accepted November 2004 Available online January 2005 Received by M. Schartl Abstract Extracellular regulated kinase (ERK5) is an unusually large member of the MAP kinase family of signaling molecules that plays an important role in cellular proliferation, differentiation and survival. Recently, three transcriptional variants of murine Erk5 were described (mErk5-a, -b and -c) that result from alternate splicing across introns and/or 2, the net effect of which is translation of a peptide that lacks the kinase domain. It has been demonstrated that expression of mErk5-b and -c impinge on the function of the full length mErk5 protein product via a dominant negative effect. Here, we report the identification of another murine Erk5 splice variant and the orthologous human transcript that arise due to alternate splicing of intron 4. Failure to splice out intron introduces a premature in-frame stop codon that directs translation of a peptide lacking the nuclear localization signal (NLS) and proline-rich region (PR). Experimental characterization demonstrated that like mERK5, mERK5-T becomes phosphorylated by co-expression with a constitutively active mMEK5 (mMEK5DD), and is able to coimmunoprecipitate with both itself and mERK5. Unlike mERK5, however, activated ERK5-T is unable to translocate from the cytoplasm to the nucleus in HeLaS3 cells, causing the retention of active mERK5 in the cytoplasm. Taken together with previous reports of domain content modification of ERK5 via alternate splicing, these observations add to the suggestion that regulation of ERK5 signaling may be mediated, at least in part, at the level of RNA processing. D 2004 Elsevier B.V. All rights reserved. Keywords: MAPK; Regulation; Signaling; Alternate splicing; Sub-cellular localization 1. Introduction Extracellular regulated kinase (ERK5) is an unusually large member of the mitogen-activated protein kinase (MAPK) family that was independently identified by two Abbreviations: T, truncated; MAPK, mitogen-activated protein kinase; ERK, extracellular regulated kinase; MEK, MAPK/ERK kinase; NLS, nuclear localization signal; PR, proline-rich domain; BMK1, big MAP kinase 1; PCR, polymerase chain reaction; RT, reverse transcriptase; RPMI, Roswell Park Memorial Institute; PAGE, polyacrylamide gel electrophoresis; HEK, human embryonic kidney; EST, expressed sequence tag. B The nucleotide sequences reported in this paper have been submitted to GenBank with accession numbers AY534740 (mErk5-T) and AY534741(hErk5-T). * Corresponding author. Tel.: +65 6586 9614; fax +65 6779 1117. E-mail address: mcbgg@imcb.a-star.edu.sg (G.R. Guy). These authors contributed equally to this work. 0378-1119/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2004.11.011 groups (Lee et al., 1995; Zhou et al., 1995). Various investigations have revealed that ERK5 plays an essential but poorly understood role in various cellular processes such as proliferation (Mulloy et al., 2003; Dong et al., 2001; Kato et al., 1998), differentiation (Reddy et al., 2002; Dinev et al., 2001) and cell survival (Zheng et al., 2004). Like all members of the MAPK family, ERK5 contains dual phosphorylation sites (TXY) and a conserved serine/threonine kinase domain. In addition, the molecule contains an oligomerization domain (OD), nuclear localization signal (NLS), and proline-rich region (PR) (Yan et al., 2001). The immediate upstream activator of ERK5 is the MAPK kinase, MEK5 (Kato et al., 1997). Upon activation, ERK5 translocates to the nucleus and phosphorylates various downstream targets including MEF2C (Kato et al., 1997) and possibly c-myc (English et al., 1998). 184 B.J. McCaw et al. / Gene 345 (2005) 183–190 The expressed portion of the mouse Erk5 gene is spread over approximately kilobases (kb) at the distal end of cytogenetic band B1.3 on chromosome 11 (NCBI Mus musculus Genome View). The major expression product from the locus is a 3-kb transcript that contains seven exons and codes for a 816 residue protein. The message is present at a relatively high level in various embryonic tissues between E11 and E17 and at a low level in most adult tissues (Regan et al., 2002). Alternate splicing of Erk5 mRNA has been reported (Yan et al., 2001). Analysis of various EST databases revealed the existence of two rare murine Erk5 (mErk5) transcript variants that arise by differential splicing of introns and 2. In both cases, inclusion of the intron introduces a premature stop codon downstream of the kinase domain coding region, with translation beginning again at an in-frame ATG located further 3V. Presumably, this is facilitated by an as-yet uncharacterized mammalian internal ribosome entry site (IRES). N-terminally truncated ERK5 translated from the alternately spliced message lacks the kinase domain and is able to inhibit the kinase activity of full length ERK5. Thus, it appears that splicing events contribute to ERK5 regulation. Here, we report the existence of another Erk5 splice variant, Erk5-T (Erk5-Truncated) that originates by alternate splicing across intron 4, the functional consequence being introduction of a premature stop codon that eliminates the nuclear translocation and proline-rich domains from the protein. Experimental observations confirmed that mERK5T is able to hetero-oligomerize with mERK5 in both active and inactive states. In addition, using immunofluorescence, we show mERK5-T remains in the cytoplasm upon stimulation and is able to retard nuclear translocation of activated mERK5 in a concentration-dependent manner. These data should help us understand how pre-translation events help regulate ERK5 signaling. 2. Materials and methods 2.1. Isolation of mouse mErk5-T cDNA Mammalian expressed sequence tags (ESTs) deposited in GenBank were searched for sequences similar to human Erk5 (GenBank accession no. BC030134) using the BLASTn program at NCBI (Altschul et al., 1997). 2.2. RNA isolation and reverse transcriptase-polymerase chain reaction (RT-PCR) Cytoplasmic total RNA was extracted from approximately 25 mg fresh tissue (or 5Â106 bone marrow cells) harvested from 6-week-old C57B/6 male mice using an RNeasy mini spin kit (Qiagen) according to the manufacturer’s instructions. First-strand cDNA was reverse transcribed from 250 ng total RNA primed with oligo dT15–18 in a 20-Al reaction containing 50 U SuperScriptII RT (Invitrogen). mErk5-T was amplified from first-strand cDNA using 5V-GCCTACTGTGCCCTATGGAG-3V (sense); 5V-TCTGTCCCAGAATCCCTGTC-3V (anti-sense) specific primers. mErk5 was amplified using the same sense primer in combination with the antisense primer 5VCGCTTCTCTTCTCGTTCTCG-3V. Primers specific for murine GAPDH were used to standardize the cDNA concentrations; 5V-TGAAGGTCGGTGTGAACGGATTTGGC-3V (sense); 5V-CATGTAGGCCATGAGGTCCACCAC-3V (anti-sense). 2.3. Construction of expression plasmids All expression constructs were generated by PCR amplification from first-strand murine bone marrow cDNA. Fragments were cloned into the HindIII and NotI sites of either pXJ40-HA or -FLAG in-frame with the N-terminal vector epitope. Oligonucleotide sets were: (a) mErk5-T: 5V-GCATAAGCTTCATCAGCGCTTCGTACAGAC-3V (sense); 5V-GCATGCGGCCGCT CTGTCCCAGAATCCCTGTC-3V (antisense); (b) mErk5: 5V-GCATAAGCTTCATCAGCGCTTCGTACAGAC-3V (sense); 5-GCATGCGGCCGCGTTTCAGGGCTCTTGGAGGT-3V (antisense); (c) mMek5: 5V-GCATAAGCTTATGCTGTGGCTGG CCCTTG-3V (sense); 5V-GCATGCGGCCGCGGTCAGTGTCCTGCTGAGGT (antisense). Full-length mErk5 clones containing intron were identified by screening colonies with oligonucleotides flanking the splice junction: 5V-GAGCTTGCTCCACCAAAAAG-3V (sense); 5V-CGCTTCTCTTCTCGTT CTCG-3V (antisense). mMek5DD (S311D, T315D) and mMek5AA (S311A, T315A) were created using single primer mutagenesis (Makarova et al., 2000), respectively; V- C A C A G C T G G T G A AT G ATATA G C C A A G G ATT A T G T T G G A A C A A A T G C T T A C - V; VCACAGCTGGTGAATGCTATAGCCAAGGCGTATGTTGGAACAAATGCTTAC-3V. 2.4. Cell culture and transfection SV40 transformed human embryonic kidney 293 T (HEK293 T) were maintained in RPMI-1640 medium (Sigma) supplemented with 10% fetal calf serum, 50 U mlÀ1 penicillin and 50 Ag mlÀ1 streptomycin. HeLa cells were cultured in Dulbecco’s Modified Eagles Medium (DMEM) supplemented with 4500 mg glucose, 10% inactivated fetal bovine serum (FBS from HyClone Laboratoriesk Logan, UT) and 1% glutamine. 2.5. Western blot analysis Cells were rinsed with phosphate-buffered saline (PBS) while attached to the plate and subsequently lysed with 0.5 B.J. McCaw et al. / Gene 345 (2005) 183–190 ml RIPA lysis buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, mM EDTA, 25 mM NaF) containing fresh 100 AM Na3VO4 and 1Â protease inhibitor cocktail (Roche). Lysates were recovered by scrapping and centrifugation before being boiled in the presence of 1Â sample buffer (50 mM Tris–HCl, pH 6.8, 10% glycerol, 2% SDS, 4% 2-mercaptoethanol, 0.02% bromophenol blue) and separated by SDS–polyacrylamide gel electrophoresis (PAGE). Fractionated proteins were electrotransferred to an activated PVDF membrane (Biorad). Protein containing membranes were blocked for 1–2 h at room temperature with 5% skim milk powder (Devondale) and then incubated for h at room temperature with a primary antibody; followed by a 1-h incubation with an appropriate horseradish peroxidase-conjugated secondary antibody. Antibody binding was visualized by chemiluminescence (ECL, Amersham Pharmacia Biotech). 2.6. Co-immunoprecipitation 293 T cell lysates (0.5 ml) were incubated with antiFLAG M2 agarose-conjugated beads (Sigma) for h at 8C. The beads were washed five times with ml lysis buffer and subsequently boiled for in 25 Al 1Â sample buffer. Five microliters of each sample was subjected to SDS–PAGE and proteins were detected by Western blotting. 2.7. Immunofluorescence Cells were seeded on poly-d-lysine (Iwakik Chiba, Japan)-coated coverslips, and transfected with 0.6–1.5 Ag of plasmid DNAs using Lipofectaminek 2000 reagent (Gibco 185 BRL) according to the manufacturer’s instructions. At 24 h post-transfection, cells were fixed with 3% paraformaldehyde for 30 at 8C and then permeabilized with 0.1% saponin (Sigma-Aldrich) in PBSCM (PBS+1 mM CaCl2+1mM MgCl2) for 20 at room temperature. For single stains, a monoclonal directed against the FLAG epitope tag (Transduction Laboratories, Lexington, KY) was used at Ag/100 Al in FDB (7% FBS+2% BSA in PBSCM); followed by Fluorescein isothiocyanate (FITC)-conjugated AffiniPure rabbit anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). For double stainings of FLAG-tagged ERK5 full-length and HA-tagged ERK5-T splice variant, HA-ERK5-T was detected using polyclonal anti-HA (Roche Molecular Biochemicals, Indianapolis, IN) and Texas Redk dye-conjugated AffiniPure goat anti-rabbit IgG (Jackson ImmunoResearch). Coverslips were mounted in Crystal Mount reagent (Biameda) and viewed by MRC1024 laser scanning confocal microscopy (Bio-Rad Laboratories) dye under 40Â oil immersion and a 1.4 numerical aperture bright-field objective. The microscopic images were processed with the aid of the LaserSharp software (Bio-Rad Laboratories) and Adobe Photoshop (Adobe System). 3. Results 3.1. Identification of murine and human intron alternatively spliced Erk5 mRNA (Erk5-T) The mammalian EST databases at NCBI were interrogated with full length human Erk5 mRNA sequence Fig. 1. Schematic representation of the human and mouse Erk5 locus showing alternate splicing across intron 4. Human Erk5/-T (top panel) and mouse Erk5/-T (bottom panel). Solid boxes indicate GenBankR sequence and nucleotide position for hErk5 (BC030134), hErk5-T EST BE300027, mErk5 (NM_011841) and mErk5-T EST (AA066727) . Shaded boxes represent sequence generated from Erk5-T RT-PCR products. A local in-frame translation of Erk5-T across intron demonstrates the introduction of a premature stop codon in both the human and mouse transcripts (putative splice acceptor and donor sites underlined). 186 B.J. McCaw et al. / Gene 345 (2005) 183–190 Fig. 2. RT-PCR analysis of mErk5 and mErk5-T in various adult C57B/6 mouse tissues. cDNA reverse transcribed from approximately 25 ng RNA (1/10 250 Ag reverse transcription reaction) was amplified by 30 cycles of PCR. Amplification products were electrophoresed through a 1.5% agarose gel and visualized by ethidium bromide staining. A 1-kb marker (NEB) served as a size marker. Template from cDNA synthesis reactions lacking reverse transcriptase were included to control for amplification from residual genomic DNA (no RT). (1) Brain, (2) bone-marrow, (3) lung, (4) heart, (5) kidney, (6) testis, (7) skeletal muscle, (8) small intestine, (9) spleen, (10) H2O control. (GenBank accession no. BC030134) using the BLASTn program (Altschul et al., 1997). Both human and mouse EST clones (human; GenBank accession no. BE300027, mouse; GenBank accession no. AA066727) were identified that contain a segment of extra internal sequence, which in both cases corresponded to intron of the Erk5 gene (Fig. 1). The mouse variant contains a 103-bp insertion at position 1516, while the alternatively spliced human transcript contains an additional 94 bp inserted at position 1497. We postulated that these EST clones represented a novel Erk5 splice variant (Erk5-T). The entire mErk5-T open reading frame (ORF) was obtained by direct sequence analysis of two overlapping PCR products amplified from murine bone marrow cDNA. These were generated by (a) coupling a sense primer within the insertion sequence with an antisense primer in the published Erk5 3V UTR; and (b) coupling an antisense primer specific for the insertion sequence with a sense primer in the mErk5 5V UTR. A similar strategy was employed to obtain sequence of the human orthologue using placental cDNA as the template. In silico translation of the mouse mErk5-T ORF identified an in-frame stop codon within the unspliced intron. Fig. 3. Detection of phospho-mERK5-T in HEK293T fibroblasts over-expressing mERK5T and mMEK5DD. (a) Schematic representation of mErk5/-T constructs. (b) Western blot. 293T cells were transfected with 0.2 Ag HA-tagged mErk5, mErk5-T or mErk5-i4us (for details, see schematic) in combination with either mMek5DD or mMek5AA. Western blotting of lysates was performed with monoclonal anti-HA (Roche). Phosphorylation of mERK5/-T was inferred by the presence of an up-shifted band (lanes 2, and 7)—see part c. (c) Western blot. Lysates made from 293T cells over-expressing HA-tagged mERK5 or mERK5-T and MEK5AA or MEK5DD were separated on a 7.5% SDS–PAGE and probed with anti-phospho Erk5 (CalBiochem). The stripped blot was probed with monoclonal anti-HA (Roche) as a loading control. B.J. McCaw et al. / Gene 345 (2005) 183–190 Similarly, retention of intron in human Erk5-T mRNA introduced an in-frame stop codon 33 bases 5V of the exon 4/5 junction via a +1-bp frame shift. Thus in both human and mouse, the alternately spliced mRNA species result in translation of an ERK5 variant that is essentially truncated after amino acid 492. The truncated protein retains the cytoplasmic targeting-, kinase- and oligomerizationdomains, while the nuclear localization signal and proline-rich domains are lost. 3.2. mErk5-T mRNA is expressed in multiple adult tissues RT-PCR analysis of RNA extracted from mouse organs was used to verify the existence of mErk5-T in the cytoplasm as well as gauge expression levels of the transcript across a range of adult tissues. This was accomplished using an oligonucleotide set specific for mErk5-T; in parallel with a set designed to amplify mErk5 cDNA spliced across intron (Fig. 2). To prevent amplification from residual genomic DNA or unspliced heteronuclear RNA, the amplicon spanned intron (2.4 kb). Compared with other tissues, Erk5 mRNA was high in bone marrow, lung, skeletal muscle and testis. Expression of mErk5-T generally correlated with Erk5 expression, although at lower levels. The only exception being the bone marrow, where a disproportionately high level of mErk5-T relative to Erk5 was consistently observed. Although an attempt was made to detect mErk5-T in tissue lysates by Western blotting, antibodies that recognize N-terminal epitopes performed poorly in our hands, failing to detect endogenous mErk5 in mouse embryonic tissue lysates (data not shown). 187 3.3. Intron of mErk5 can be alternately spliced, and both products are potential substrates of mMEK5 To assess whether intron has the potential to be alternately spliced, a construct was made that contained 5V HA-tagged mErk5 cDNA unspliced with respect to intron (mErk5-i4us, Fig. 3a). The construct was transfected into 293T cells in parallel with HA-tagged mErk5 and mErk5-T alone. Whole cell lysates were separated on a 7.5% PAGE and probed with monoclonal anti-HA. A 110-kDa immunoreactive band was detected in lysates made from cells expressing HA-mERK5, and a 60-kDa band was detected in lysates made from HA-mERK5-T transfected cells (Fig. 3, lanes and 3). Both the 110- and 60-kDa (albeit at a lower intensity) bands were detected in lysates made from cells transfected with mErk5-i4us, demonstrating that the (overexpressed) alternatively spliced mErk5-T mRNA can be exported from the nucleus to the cytoplasm and translated. In order to investigate whether mERK5-T can be phosphorylated by its upstream kinase (mMEK5), the three mERK5 constructs (mErk5, mErk5-T and mErk5-i4us) were co-transfected with either a constitutively active mMEK5 (mMEK5DD), or a dominant negative mMEK5 (mMEKAA). Phosphorylation of mERK5 can be inferred by a shift in mobility of the protein through a 7.5% SDS–PAGE gel (Kato et al., 1998). A shift in both mERK5 and mERK5T mobility was consistently seen when co-transfected with mMEK5DD relative to mMEK5AA (Fig. 3, compare lanes with 2, with and with 6) indicative of mERK5/-T phosphorylation. The phosphorylation status of mERK5/-T was confirmed by immunoreactivity of 110- and 60-kDa bands with an anti-phospho Erk5 antibody (Fig. 3a). It is Fig. 4. mERK5-T remains in the cytoplasm upon stimulation. HelaS3 cells were co-transfected with Ag FLAG-mErk5 full-length and 0.5 Ag of either vector control (A), MEKKDD (B) MEKKAA (C); or Ag FLAG-mErk5-T and 0.5 Ag of either vector control (D), MEKKDD (E) MEKKAA (F). Cells were then fixed, permeabilized and stained for FLAG-tagged ERK5 using an anti-FLAG monoclonal followed by fluorescein isothiocyanate (FITC)-conjugated AffiniPure rabbit anti-mouse IgG (green). 188 B.J. McCaw et al. / Gene 345 (2005) 183–190 the NLS of ERK5, its cellular localization in response to stimulation warranted experimental investigation. Immunofluorescence of HeLaS3 cells over-expressing HA-tagged mErk5-T was used to monitor the cellular localization of mERK5-T co-expressed with either mMEKAA or mMEK5DD. The same experiment was performed with mErk5 as a positive control. As expected, non-phosphorylated mERK5-T and ERK5 localized to the cytoplasm, while only stimulated mERK5 was able to translocate to the nucleus (Fig. 4). Stimulated mERK5-T failed to enter the nucleus and displayed a sub-cellular distribution indistinguishable from unstimulated mERK5-T. These data imply that a functional consequence of alternate splicing across intron is possibly retention of activated mERK5 in the cytoplasm. Fig. 5. mERK5-T associates with itself and mERK5-T. HEK293T cells were transfected with various combinations of differentially tagged (either FLAG or HA) mErk5 and mErk5-T in the presence of mMek5AA or mMek5DD. Co-immunoprecipitation was performed with anti-FLAG M2 conjugated agarose beads (Sigma), and Western blot was performed with anti-HA (Roche). Expression of constructs was confirmed by Western blot of whole cell lysates (WCL) with either anti-HA and anti-FLAG. interesting to note however, that the level of mERK5-T relative to mERK5 in lysates made from cells expressing mErk4-i4us was much reduced in the presence of mMEK5DD (relative to mMEK5AA). 3.4. mERK5-T remains in the cytoplasm upon stimulation In line with other members of the MAPK family, ERK5 translocates from the cytoplasm to the nucleus upon stimulation (Kato et al., 1997). However, as mERK5-T lacks 3.5. mERK5-T can heterodimerize with ERK5 mERK5 exists as a homo-oligomer in a manner that is independent of the molecules’ phosphorylation status (Yan et al., 2001). As the oligomerization domain is retained in mERK5-T, its potential to associate with both itself and mERK5 was assessed in co-immunoprecipitation experiments. To accomplish this, FLAG-mERK5 was coexpressed with HA-mERK5-T and either mMEK5AA or mMEK5DD in HEK293T fibroblasts. As can be seen in Fig. 5, immunoprecipitation of phosphorylated and non-phosphorylated FLAG-ERK5 brought down HA-tagged mERK5-T, indicating that mERK5/mERK-T heterodimers can exist in both a stimulated and unstimulated state. The potential for homo-oligomerization of mERK5-T was also shown by successful co-precipitation of HA-mERK5-T with FLAG- ERK5-T. Fig. 6. mERK5-T inhibits nuclear translocation of phospho-mERK5. HelaS3 cells were triply transfected with 0.2 Ag FLAG-mErk5 full-length, 0.2 Ag HAmErk5-T and 0.2 Ag of either MEKKDD (a, b) or MEKKAA (c, d); or 0.2 Ag FLAG-Erk5 full-length, 1.0 Ag HA-mErk5-T and 0.2 Ag of either MEKKDD (e, f) or MEKKAA (g, h). Cells were then fixed, permeabilized and stained for FLAG-ERK5 using an anti-FLAG monoclonal (a, c, e, g) and fluorescein isothiocyanate (FITC)-conjugated AffiniPure rabbit anti-mouse IgG (green). HA-tagged mERK5-T was visualized with an anti-HA polyclonal (b, d, f, h) followed by Texas RedR dye-conjugated AffiniPure goat anti-rabbit IgG (red). B.J. McCaw et al. / Gene 345 (2005) 183–190 3.6. Effect of mERK5-T on phospho-mERK nuclear localization Translocation of phosphorylated mERK5 from the cytoplasm to the nucleus is facilitated by the presence of a NLS. As mERK5-T and mERK5 differ in this respect, and potentially remain complexed upon phosphorylation, it was postulated that co-expression of mErk5-T may inhibit nuclear translocation of phospho-mERK5. To explore this possibility, FLAG-tagged mErk5 was cloned into pIRES (3V MCS) along with either mMek5AA or mMek5DD (5VMCS). The mErk5 and mMek5 constructs were then co-transfected with HA-mErk5-T or empty pXJ40HA into HeLaS3 fibroblasts at various ratios; Ag mErk5:1/2/5 Ag mErk5T. The sub-cellular localization of mERK5 and mERK5-T were visualized by confocal immunofluorescence (Fig. 6). As seen previously, mERK5 localized to the cytoplasm when expressed from bi-cistronic message with mMek5AA, but translocated to the nucleus when expressed bi-cistronicly with mMek5DD. Co-expression of mErk5-T however, appeared to inhibit the translocation of the full length molecule in a concentration dependent manner. At a DNA transfection ratio of 1:1, the observed inhibition was evident but slight, whereas at 1:2 (not shown) and 1:5 ratios, the inhibition seemed complete. 4. Discussion Here, we report the identification and initial characterization of an alternative mammalian Erk5 splice variant (mErk5-T) that retains the intervening sequence between exons and (intron 4). Alternative splicing across intron introduces a stop codon in the mouse and orthologous human transcripts, the predicted effect being translation of an ERK5 variant (ERK5-T) that is truncated at the Cterminus and loses the NLS and PR domain. These observations complement prior reports of alternate splicing across mErk5 introns and 2, leading to expression of an ERK5 variant that lacks the kinase domain (Yan et al., 2001). The expression pattern of mErk5-T in adult tissues correlates with that of mErk5, albeit at significantly lower levels with possible relative enrichment of mErk5-T in the total bone marrow transcriptome. These data however, not exclude the possibility that mErk5-T is expressed at a relatively high level in specific cells under certain conditions. While in situ hybridization can be used to address this point, the subtle sequence difference between the mErk5 and mErk5-T transcripts (+103 bp) may be insufficient to distinguish the two splice forms. As far as we are aware, this is the first example of an Erk5 tissue expression analysis that includes the bonemarrow. Interestingly, bone marrow is one of the four tissues (testis, skeletal muscle, lung and bone marrow) that display an enrichment of the Erk5 (and Erk5-T) transcript. This 189 observation is in-line with previously published signaling data that suggests ERK5 is a down-stream component of certain cytokine receptors involved in hematopoiesis, e.g. G-CSFR (Dong et al., 2001). Consistent with retention of the MEK5 phosphorylation targets (T219, Y221), mERK5-T can be phosphorylated by constitutively active MEK5DD. Preliminary data also indicates that phosphorylation of mERK5-T seems to play a determinate role in the ratio of mERK5/mERK5-T in cells over-expressing an intron unspliced transcript. Whether this results from regulation at the level of mRNA splicing or events further down-stream (mRNA/protein degradation) remains unclear. Retention of the oligomerization domain within mERK5T most likely explains the ability of mERK5-T to associate with both itself and mERK5 in active and inactive states. Similarly, the failure of mERK5-T to translocate from the cytoplasm to the nucleus upon stimulation is consistent with loss of the C-terminal NLS. The most profound observation made of mERK5-T though, is the inhibitory effect on nuclear translocation of activated mERK5. These data imply that in a physiological context it is possible that expression of mERK5-T serves to attenuate nuclear signaling of mERK5, and/or may represent a mechanism of maintaining mERK5 signaling in the cytoplasm through as yet unidentified downstream cytosolic targets. Although not elicited by alternate splicing and domain content manipulation, it is well established that ERK1/2 signaling elicits distinct cellular processes through differential compartmentalization of phospho-ERK. Sustained ERK1/2 activation leads to differentiation through nuclear localization (and signaling) of the active molecule, whereas transient activation stimulates proliferation via cytosolic ERK1/2 signaling (Marshall, 1995). It has been observed in conventional knock-out studies that mERK5-deficient mice develop angiogenic defects and die in utero (Sohn et al., 2002). In a separate study, conditional disruption of mErk5 led to loss of vascular integrity and subsequent endothelial failure (Hayashi et al., 2004). In both cases, transcriptional dysregulation of genes downstream of mERK5 signaling was suggested to be responsible for the observed phenotypes. Since mERK5-T inhibits nuclear translocation of mERK5, this may represent a novel regulatory mechanism of such physiological activities. It is possible that changing ratios of mERK5-T/ mERK5 modulate the levels of phosphorylated mERK5 in the nucleus, which subsequently affects transcriptional activation of genes involved in these processes. This implies that such physiological processes may not be regulated simply by the presence or absence of mERK5, but the degree of nuclear Erk5 activity, where moderate changes in the availability of mERK5 to transcription factors may affect physiological outcome. In summary, data presented in this manuscript describes a novel human and mouse ERK5 splice variant that lacks a functional NLS and as such is unable to enter the nucleus 190 B.J. McCaw et al. / Gene 345 (2005) 183–190 upon activation. Taken together with prior reports of mERK5 functional domain architecture modification by alternative splicing, these data add to the suggestion that regulation of ERK5 signaling could, at least in part, be carried out at the level of mRNA processing. References Altschul, S.F., et al., 1997. 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[...]... activity of GTPases is very inefficient, thus necessitating the assistance of GTPase activating proteins (GAPs) such as RasGAP The relative amounts of the two states of Ras, and hence its activity, depends on the balance between GEFs and GAPs There are three main isoforms of Ras, namely H-, K- and N-Ras They share approximately 90% similarity over most of their sequences, and are most divergent in the last... present in the extracellular and cytoplasmic regions of different classes of RTKs 3 1.2.1 Activation of RTKs In unstimulated cells, RTKs often exist as monomers in the cell membrane Ligand binding induces the dimerization of the receptors, either because a single ligand can bind to and stabilize two molecules of the receptor, or because the ligand itself is a dimer (Schlessinger, 2000) This brings the cytoplasmic... domains of the RTKs into close proximity, such that the Tyr residues within the activation loop can be phosphorylated This stabilizes the conformation of the catalytic domain of the RTK, and allows its full activation The RTK then phosphorylates several Tyr residues either within its own sequence, as in the case of the EGF receptor (EGFR), or in a closely-associated docker protein, as in the case of the. .. its activation loop Further analysis showed that Spry2, PKCδ and PKD1 form a trimeric complex In order for Spry2 to interact with PKCδ, PKD1 and PKCδ must first bind to each other The role that Spry2 plays within this complex is to lock the interaction between PKCδ and PKD1, and block the transfer of a phosphate XIV group from PKCδ to PKD1 The interaction between Spry2 and PKCδ therefore effectively... connectors to other downstream signaling molecules by interacting with them through other domains, thus recruiting them to the active complex at the membrane This interaction process results in the activation of other signaling molecules, either due to membrane anchoring, Tyr phosphorylation by the RTK, or both (Schlessinger, 2000) Recruitment of each of these adaptors and enzymes allows activation of a limited... recruitment and activation of PLCγ allows the hydrolysis of its substrate phosphatidyl inositol 4,5bisphosphate [PtdIns(4,5)P2], ultimately resulting in the release of two second messengers, inositol 1,4,5-trisphophate [Ins(1,4,5)P3] and subsequently Ca2+, and diacylglycerol (DAG) On the other hand, activation of the phospholipid kinase PI3K leads to the phosphorylation of PtdIns(4,5)P2 and induction of the. .. protein kinase Cδ (PKCδ) was further identified to be a FGF stimulationdependent interacting partner of Spry2 The interaction between Spry2 and PKCδ was also found to be both specific and direct, and it depends on the conformation of the two proteins The binding of Spry2 and PKCδ does not inhibit phosphorylation or activation of PKCδ Instead, it inhibits the phosphorylation of a PKCδ substrate, protein... Additionally, the interaction between Ras and Raf is also believed to release intramolecular inhibitory interactions arising from the N-terminal regulatory domain of Raf (Wellbrock et al., 2004) Displacement of 14-3-3 from Raf follows Ras binding, as a result of the dephosphorylation of Raf by PP2A and/ or PP1 (Abraham et al., 2000; Jaumot and Hancock, 2001) Phosphorylation then occurs on Ser338 and Tyr341... of Raf for Ras binding Signal propagation between Ras and Raf would therefore be reduced Results from this study indicate that the expression of Spry2 increases the interaction between active Ras and RIN1 Reports have suggested that the kinase activity of PKCδ is required for the invasive potential of prostate cancer cells Cell invasion assays in this study show that by inhibiting phosphorylation of. .. Ser and Thr residues, of which one forms a Ser phosphorylation site which is part of the consensus motif for the interaction with the adaptor protein 14-3-3 (S259 in Raf1) The N-terminal half of Raf is collectively known as the regulatory domain, and is responsible for holding the protein in an inactive conformation in unstimulated cells The C-terminal CR3 encompasses the catalytic kinase domain of . THE MECHANISM OF ACTION OF SPROUTY2: CHARACTERIZATION OF THE INTERACTION BETWEEN SPROUTY2 AND PKCδ CHOW SOAH YEE B.Sc. (Hons) National University of Singapore A THESIS. alternatives for the mechanism of action by Spry2 121 3.3.7 Increasing levels of PKD1 enhances the interaction between PKCδ and Spry2 122 3.3.8 Verification that PKD1 increases the interaction between. representation of the primary structure of Raf 15 1.6.2 The structure and isoforms of Spry 27 1.7 Schematic representation of PLCγ signaling 39 1.7.1 Schematic representation of the primary structure of