Biochemical and genetic analysis of the adaptor protein SH3P7 [Elektronische Ressource] : insights from a newly generated knockout mouse / vorgelegt von Sabine Connert

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BIOCHEMICAL AND GENETIC ANALYSIS OF THEADAPTOR PROTEIN SH3P7:INSIGHTS FROM A NEWLY GENERATED KNOCKOUTMOUSEInaugural Dissertationzur Erlangung der Doktorwürdeder Fakultät für Biologieder Albert-Ludwigs-Universität Freiburgim Breisgauvorgelegt vonSabine Connertaus Mediasch in SiebenbürgenFreiburg 2003Dekan der Fakultät Prof.Dr.Hans KleinigPromotionsvositzender Prof.Dr.Karl-Friedrich FischbachBetreuer der Arbeit Prof.Dr.Michael RethLeiter der Arbeit Prof.Dr.Jürgen WienandsKorreferent PD Dr.Hermann EibelDritter Prüfer Prof.Dr.Hans Ulrich WeltzienTag der Verkündigung des Prüfungsergebnisses: 21.Mai 2003Die vorliegende Arbeit wurde von Oktober 1999 bis Januar 2002 am Max-Planck-Institut für Immunbiologie in Freiburg und von Februar 2002 bis Januar 2003 an derUniversität-Bielefeld durchgeführt. Die Arbeit wurde unterstützt durch dasMinisterium für Wissenschaft, Forschung und Kunst des Landes Baden-Württemberg(Forschungsschwerpunkt "Immundefizienzen") und durch die DeutscheForschungsgemeinschaft (SFB 549).Index1 SUMMARY.........................................................................................................................................52 ZUSAMMENFASSUNG..................................................................................................................63 INTRODUCTION73.1 BCR RECEPTOR SIGNALING .......................................................................................................83.
Publié le : mercredi 1 janvier 2003
Lecture(s) : 15
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Source : WWW.FREIDOK.UNI-FREIBURG.DE/VOLLTEXTE/724/PDF/DISSERTATION.PDF
Nombre de pages : 123
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BIOCHEMICAL AND GENETIC ANALYSIS OF THE
ADAPTOR PROTEIN SH3P7:
INSIGHTS FROM A NEWLY GENERATED KNOCKOUT
MOUSE
Inaugural Dissertation
zur Erlangung der Doktorwürde
der Fakultät für Biologie
der Albert-Ludwigs-Universität Freiburg
im Breisgau
vorgelegt von
Sabine Connert
aus Mediasch in Siebenbürgen
Freiburg 2003Dekan der Fakultät Prof.Dr.Hans Kleinig
Promotionsvositzender Prof.Dr.Karl-Friedrich Fischbach
Betreuer der Arbeit Prof.Dr.Michael Reth
Leiter der Arbeit Prof.Dr.Jürgen Wienands
Korreferent PD Dr.Hermann Eibel
Dritter Prüfer Prof.Dr.Hans Ulrich Weltzien
Tag der Verkündigung des Prüfungsergebnisses: 21.Mai 2003
Die vorliegende Arbeit wurde von Oktober 1999 bis Januar 2002 am Max-Planck-
Institut für Immunbiologie in Freiburg und von Februar 2002 bis Januar 2003 an der
Universität-Bielefeld durchgeführt. Die Arbeit wurde unterstützt durch das
Ministerium für Wissenschaft, Forschung und Kunst des Landes Baden-Württemberg
(Forschungsschwerpunkt "Immundefizienzen") und durch die Deutsche
Forschungsgemeinschaft (SFB 549).Index
1 SUMMARY.........................................................................................................................................5
2 ZUSAMMENFASSUNG..................................................................................................................6
3 INTRODUCTION7
3.1 BCR RECEPTOR SIGNALING .......................................................................................................8
3.2 ENDOCYTOSIS OF THE BCR .....................................................................................................11
3.3 THE ACTIN-CYTOSKELETON AND ITS ROLE IN SIGNALING AND ENDOCYTOSIS.......................13
3.4 THE ADAPTOR PROTEIN SH3P7................................................................................................14
3.5 GENE TARGETING AND THE GENERATION OF KNOCKOUT MICE ..............................................17
4 METHODS.........21
4.1 RECOMBINANT DNA TECHNIQUES..........................................................................................21
4.1.1 Generation and transformation of competent bacteria................................................21
4.1.2 Production and purification of GST fusion proteins ....................................................22
4.1.3 Plasmid DNA amplification...........................................................................................23
4.1.4 Measurement of DNA concentrations...........................................................................24
4.1.5 Restriction digest and ligation of DNA.........................................................................24
4.1.6 Separation of DNA fragments on agarose gels ............................................................24
4.1.7 Purification of DNA from agarose gels ........................................................................24
4.1.8 Preparation of genomic DNA........................................................................................25
4.1.9 Amplification of DNA fragments from genomic DNA..................................................25
4.1.10 Genotyping of mice ........................................................................................................25
4.2 SOUTHERN BLOTTING AND HYBRIDIZATION............................................................................26
4.2.1 Southern blotting............................................................................................................26
324.2.2 Hybridization with P-labelled probes26
4.3 DNA SEQUENCING ...................................................................................................................27
4.4 CELL CULTURE OF MAMMALIAN CELLS ...................................................................................28
4.4.1 Cell lines.........................................................................................................................28
4.5 GENERATION OF KNOCKOUT MICE FROM ES CELLS................................................................29
4.5.1 General principle..29
4.5.2 Preparation of gelatin-coated tissue culture plates for ES cell culture ......................29
4.5.3 Preparation of mouse embryonic fibroblast and feeder layers for ES cell culture.....30
4.5.4 Cultivation of mouse ES cells........................................................................................31
4.5.5 Transfection of mouse ES cells......................................................................................31
4.5.6 Picking of ES cell clones ...............................................................................................32
4.5.7 Screening of ES cells for homologous recombinants ...................................................32
4.5.8 Freezing of ES cell clones in 96-well plates.................................................................32
4.5.9 Freezing of ES cell clones in single vials......................................................................33
4.5.10 Thawing ES cell clones from 96-well plates33
4.6 MOUSE STRAINS .......................................................................................................................33
4.7 FLOW CYTOMETRY...................................................................................................................33
4.7.1 Flow cytometric analysis33
4.7.2 Preparation of cell suspensions of different mouse organs .........................................34
4.7.3 Internalization assay......................................................................................................34
4.8 PROTEIN BIOCHEMISTRY ..........................................................................................................35
4.8.1 Stimulation of cells and preparation of cell lysates .....................................................35
4.8.2 Immuno-purification......................................................................................................35
4.8.3 SDS-PAGE and immunoblotting ...................................................................................35
4.9 HISTOCHEMISTRY.....................................................................................................................36
4.9.1 Preparation of cryo-sections.........................................................................................36
4.9.2 Staining of cryo-sections ...............................................................................................37
4.10 VARIOUS BUFFER COMPOSITIONS ............................................................................................37
4.11 DATABASE SOURCES AND ONLINE SEQUENCE SEARCH TOOLS................................................37
4.12 TEXT, IMAGE AND GRAPHICS PROCESSING SOFTWARE............................................................38
5 RESULTS.........................................................................................................................................39
5.1 SH3P7 BINDS TO HIP1R...........................................................................................................39
1Index
5.2 SH3P7 ASSOCIATES WITH SEVERAL COMPONENTS OF THE ENDOCYTIC MACHINERY ............41
5.3 B CELL ANTIGEN RECEPTOR STIMULATION REDUCES HIP1R-BINDING OF SH3P7 .................44
5.4 GENERATION OF A NEW MOUSE MODEL: CLONING AND CHARACTERIZATION OF SH3P7 GENE
FROM MICE...............................................................................................................................................49
5.5 GENERATION OF SH3P7 KNOCKOUT MICE................................................................................57
5.6 DELETION OF SH3P7 IN MICE IS NOT LETHAL DESPITE ITS WIDE EXPRESSION .......................64
-/-5.7 BCR INTERNALIZATION IS ACCELERATED IN SH3P7 MICE...................................................65
-/- +/-
5.8 MALE SH3P7 AND SH3P7 DEVELOP A FATAL DISEASE......................................................67
5.9 B CELL DEVELOPMENT OR MATURATION MIGHT BE IMPAIRED IN MALE SICKENED MICE ......74
5.10 PARALYSIS OF MALE SH3P7 KNOCKOUT MICE IS LIKELY CAUSED BY A NEUROLOGICAL THAN
A MUSCULAR DEFECT ..............................................................................................................................77
6 DISCUSSION....78
6.1 SH3P7/HIP1R INTERACTION IS THE FIRST STRUCTURAL LINK BETWEEN BCR STIMULATION,
THE ACTIN-CYTOSKELETON AND ENDOCYTOSIS.....................................................................................78
6.2 INTERACTION PARTNERS OF SH3P7: HOMOLOGIES FROM YEAST TO MAMMALS ...................82
6.3 THE SH3P7 KNOCKOUT MOUSE: A NEW ANIMAL MODEL TO STUDY SH3P7 FUNCTION.........89
6.4 SH3P7 IS IMPLICATED IN REGULATION OF BCR INTERNALIZATION ......................................90
6.5 ADULT MALE KNOCKOUT MICE AS WELL AS HETEROZYGOUS MICE DEVELOP A FATAL
DISEASE ..................................................................................................................................................92
6.6 PROTEINS WHICH MIGHT COMPENSATE FOR THE LACK OF SH3P7..........................................96
6.7 SIMILARITIES BETWEEN THE LOWE SYNDROME AND THE SH3P7 KNOCKOUT ........................98
6.8 OUTLOOK...99
7 REFERENCES..............................................................................................................................100
8 APPENDIX......110
8.1 AMPLIFIED GENOMIC DNA FRAGMENTS AND RESULTING PLASMIDS...................................110
8.2 OLIGONUCLEOTIDES...............................................................................................................111
8.3 CONSTRUCTION OF THE SH3P7 DELETION VECTOR................................................................115
8.3.1 Plasmid maps of intermediate constructs ...................................................................115
8.3.2 Construction strategy for the sh3p7 deletion vector ..................................................116
8.3.3 Plasmid map of the KO-vector for deletion of sh3p7.................................................118
8.4 PROBES FOR SOUTHERN BLOTTING ........................................................................................119
8.5 AMINOACIDS AND THE NUCLEIC ACID TRIPLETT CODE .........................................................120
2Abbreviations
Abbreviations
°C degree Celsius
aa aminoacids
Ab antibody
amp ampicillin
APC antigen presenting cell
BCR B cell antigen receptor
BGH bovine growth hormone
BTKBruton’s tyrosine kinase
cDNA complementary DNA
CHC clathrin heavy chain
DMSO dimethylsulfoxide
dNTP desoxy nucleotide trisphosphate
EF embryonic fibroblast
ES cell embryonic stem cell
EST expressed sequence tag
Ex exon
FCS fetal calf serum
FITC fluorescein
G418 geneticin
gDNA genomic DNA
h hour(s)
Ig immunoglobulin
In intron
IP immuno-purification (affinity purification with Ab)
IP inositol 1,3,4,5-tetrakisphoshate4
ITAM immunoreceptor tyrosine based activation motif
Kana kanamycin
LIF leukemia inhibitory factor
min minute(s)
neo neomycin
3Abbreviations
neo-cassette neomycin resistance cassette
NPTII neomycin phosphotransferase II
ORF open reading frame
PAGE polyacrylamide gel electrophoresis
PBS phosphate buffered saline
PCR polymerase chain reaction
PCR
PE phycoerythrin
PerCP peridinin chlorophyll protein
PFA paraformaldehyde
PH plecstrin homology
PI(3,4,5)P phosphatidyl-inositol 3,4,5-trisphosphate3
PI(4,5)P phosphatidyl-inositol 4,5-bisphosphate2
PKC protein kinase C
PLC phospho lipase C
PTKprotein tyrosine kinase
RNAi RNA interference
s second(s)
SDS sodium dodecyl sulfate
SH2/SH3 src homology 2/3
TCR T cell antigen receptor
Tf transferrin
Tf R transferrin receptor
TKthymidine kinase
4Summary
1 Summary
SH3P7 (mAbp1/Hip55) is a widely expressed actin-binding adaptor protein
identified as a substrate for protein tyrosine kinase (PTK) after BCR activation. In
addition to two protein-binding modules specific for filamentous actin, SH3P7 also
contains an SH3 domain. The biochemical studies presented here, show that SH3P7
interacts via this domain with key components of endocytosis in B cells. SH3P7
binding to Hip1R, a huntingtin family member and new component of clathrin-
coated vesicles, is characterized in greater detail. In resting B cells, Hip1R is
constitutively bound to SH3P7. Stimulation of B cells with BCR-crosslinking-
antibody or pervanadate-treatment, abolishes the interaction in a time-dependent
manner. Thus the SH3P7/Hip1R interaction might be a structural link between BCR
activation, the actin-cytoskeleton and endocytosis. To inactivate the mouse sh3p7
gene, the gene was first cloned and characterized. This allowed the construction of a
deletion vector, which was used to target the sh3p7 gene in embryonic stem cells.
-/-Sh3p7 mice were successfully produced and are viable and fertile. Initial
-/-experiments with sh3p7 B cells indicate a role of SH3P7 as inhibitor of BCR
-/- +/-internalization. Male sh3p7 and sh3p7 mice of this strain develop splenomegaly
and a progressive and ultimately fatal disease at 3-4 months of age. Initial FACS-
analysis of the cellular composition of the spleen show drastic reduction of T1 B
cells which might be interpreted as defect in B cell development or maturation.
-/-Symptoms such as progressive paralysis implicate a neuronal disease in male sh3p7
+/-and sh3p7 mice. Future detailed studies of the sh3p7 knockout might give insight
into related human diseases.
In summary provides this work evidence for a function of SH3P7 in BCR
endocytosis and shows the result of the generation and initial analysis of a new
knockout mouse.
5Zusammenfassung
2 Zusammenfassung
SH3P7 (mAbp1/Hip55) ist ein nahezu ubiquitär exprimiertes, Aktin-bindendes
Adaptorprotein, welches als Substrat von Proteintyrosinkinasen in stimulierten B
Zellen identifiziert wurde. SH3P7 besitzt ausser zwei F-Aktin-bindenden
Interaktionsdomänen, unter anderem eine SH3-Domäne. Im Folgenden gezeigte
Ergebnisse biochemischer Experimente belegen die Assoziationen der SH3 Domäne
zu Komponenten der Endozytose in B Zellen. Die konstitutive Bindung zwischen
SH3P7 und Hip1R, die nach BCR-Kreuzvernetzung vermindert wird, könnte ein
Bindeglied zwischen BCR-Aktivierung, Aktinzytoskelett und Endozytose darstellen.
Um eine sh3p7-Knockout-Maus zu generieren, wurde zunächst das SH3P7
kodierende Gen kloniert und charakterisiert, ein Knockout Vektor konstruiert und
damit Bereiche eines sh3p7-Allels in embryonalen Stammzellen deletiert. Die daraus
-/-erfolgreich hergestellten sh3p7 Mäuse sind lebensfähig und fruchtbar. Erste
Analysen von B Zellen der sh3p7-Knockout Mäuse deuten auf eine inhibitorische
Funktion des Adaptors bei der BCR-Internalisierung hin. Knockout-Männchen und
solche heterozygoten Genotyps entwickeln nach einigen Monaten Splenomegalie
und eine schnell fortschreitende tödliche Krankheit. FACS-Analysen der
Zellpopulationen vergrösserter Milzen ergab ein deutliche Verminderung von T1 B
Zellen, was auf einen Defekt in der B Zellentwicklung oder Reifung schließen lassen
-/- -/+könnte. Lähmungserscheinungen bei sh3p7 und sh3p7 Männchen sind
wahrscheinlich auf einen neuronalen Defekt zurückzuführen. Erforschung der
molekularen Ursachen könnten Einsichten Erkenntnisse zu ähnliche Krankheiten des
Menschen bringen.
Zusammenfassend lässt diese Arbeit auf eine Funktion von SH3P7 in BCR
Endozytose schließen und präsentiert eine neue Knockout Maus.
6Introduction
3 Introduction
The immune system is a unique system capable of recognizing, and combating
foreign invaders of the body. The innate immune system provides already unspecific
protection, whereas the acquired immune system has additional characteristic
attributes. In a fascinating and not yet completely understood fashion it is able to
distinguish between non-self and self and to specifically react against tremendous
numbers of foreign antigenic structures. Remarkable is not only the great diversity of
recognition molecules on cells of the immune system, but also the capacity to
memorize and react faster and more specificly in a secondary memory response. To
perform its functions, a large and complex network of communication between its
components is necessary, comparable in its complexity perhaps only with the
neuronal system.
An effective immune response involves mainly two cell types: Classical antigen
presenting cells (APCs), such as macrophages or dendritic cells, and lymphocytes,
which can be separated in T lymphocytes (T cells) and B lymphocytes (B cells). T
and B cells arise from hematopoetic stem cells in the bone marrow. T cells require
the thymus for further development as additional primary lymphoid organ. During
their development, they undergo high selection pressure whereby the majority of T
and B cells is eliminated. This selection is centered on the successive rearrangement,
expression and specificity of their antigen receptors (Rajewsky, 1996). Mature T and
B cells circulate through the body via the blood stream and the lymph. They home to
secondary lymphoid organs such as lymph nodes and spleen, encounter foreign
antigen via their T cell antigen receptor (TCR) or their B cell antigen receptor
(BCR), respectively, and mount specific immune responses. T and B cells are able to
communicate with each or with other APCs via direct cell-cell contacts through
surface molecules and via the secretion of cytokines. The structure and amount of
antigen, the affinity and the avidity of ligand-binding, determine the kind of signal
(stimulatory or inhibitory) which is transduced into complex intracellular signaling
cascades and into the adequate cell response (Batista and Neuberger, 1998). The
understanding of intracellular signaling in addition to knowledge about extracellular
signaling, is an important prerequisite for complete understanding of the immune
system. Amplification and diversification of signals in the cytoplasm is mediated
7Introduction
through interaction of different effector molecules, catalyzing enzymes and structural
components or adaptors (Reth and Wienands, 1997).
3.1 BCR receptor signaling
The BCR is a multi-subunit transmembrane receptor in B lymphocytes. It contains
two membrane-bound disulfid-linked immunoglobulin (Ig) heavy chains, each linked
with one Ig light chain, and an Igα/Igβ heterodimer (figure 1) (Campbell and
Cambier, 1990; Hombach et al., 1990a; Hombach et al., 1990b; Matsuuchi and Gold,
2001; Schamel and Reth, 2000). Together, these subunits perform a bivalent
function: BCR signal transduction after receptor crosslinking through antigen-
binding, and uptake of the bound antigen for processing and presentation to T
lymphocytes (figure2) (Reth and Wienands, 1997; Wagle et al., 1998). Both
functions are essential for B cell activation and mounting a specific immune response
against the recognized antigen.
Figure 1: The B cell antigen receptor.
The very early events after BCR crosslinking are protein tyrosine phosphorylation of
its Igα/Igβ subunits, activation of the proteine tyrosine kinase (PTK) Syk and
subsequent phosphorylation of Slp65 (BLNK/BASH) a key adaptor in BCR
signaling (Hutchcroft et al., 1991; Hutchcroft et al., 1992; Kurosaki, 1999; Wienands
et al., 1998). Phosphorylation of tyrosines within immunoreceptor tyrosine based
8

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