Universität zu Köln

Shekar, Sunil (2005). Characterization of Cyclase Associated Protein homologue, CAP2. PhD thesis, Universität zu Köln.

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Abstract

Cyclase associated protein (CAP) is a bifunctional protein having an N-terminal adenylyl cyclase binding domain and a C-terminal actin-binding domain. In-between these two domains there is a proline rich SH3 domain presumably involved in the localization of CAP. CAP is expressed widely and has been found in yeast, Dictyostelium, Drosophila, rat, mouse and humans. Two homologues of CAP have been identified in higher eukaryotes, CAP1 and CAP2. Although CAP proteins have been studied for more than a decade and are present in all organisms, many questions remain about the mechanisms of CAP function. The roles of mammalian CAP2 proteins have not been investigated extensively. In our study we showed that CAP2 has 2 transcripts of 3 and 3.5 kb unlike the ubiquitously expressed CAP1, homologue of CAP2 in mice. In contrast to CAP1, CAP2 is expressed in a tissue specific manner. We found that CAP2 is present in relatively moderate levels in brain, heart and skeletal muscle and lower levels in skin. Furthermore, we investigated the expression pattern in more detail in these tissues and found that CAP2 is present in all parts of the adult brain but is detected only in the cerebellum and brain stem in the newborn mice and expressed only in the cerebellar region of the brain in embryos of E16. In brain CAP2 was expressed uniformly in the cortex and also found in the Purkinje cells and in the myelinated axons of the white matter. In skeletal muscle we observed a striated pattern..CAP2 was strongly expressed in the heart where it was present in cardiomyocytes, endothelial cells and the capillary wall of the blood vessels. We observed a striated pattern, which correlated with an association of CAP2 with the M-bands. In the skin CAP2 was expressed in the basal epidermal layers and in the hair follicle region and was found in the sebaceous glands. CAP2 was also expressed in the keratinocytes in skin. Immunofluorescence studies with PAM212, a mouse keratinocyte cell line, and primary mouse keratinocytes revealed that CAP2 was surprisingly localized in the nucleus. Cell fractionation experiments performed with PAM212 cells confirmed the presence of CAP2 in the nucleus. In the case of primary mouse keratinocytes CAP2 was also localised to the nucleus apart from its presence in the cytosol. In contrast in human keratinocytes CAP2 localised to the cell cortex and present in patches. Immunofluorescence studies and experiments with drugs affecting the cytoskeleton revealed that CAP2 was partially associated with F-actin and the microtubules. The nuclear localization was dependent on the microtubular cytoskeletal network and independent of the actin cytoskeleton. Overexpression studies in HEK293 cells using EGFP and Myc fusion proteins showed that CAP2 is cytosolic and was associated with lamellipodia. Unlike our overexpression studies, the immunofluorescence studies with HL-1, a cardiomyocyte cell line, and with primary cardiomyocytes of embryonic stages E17 and E19 showed that CAP2 was localized to the nucleus as in the PAM212 cells. In cardiomyocytes, CAP2 colocalised partially with Troponin I. In order to investigate the interaction with its homologue CAP1, cotransfection of GFP-CAP1, Myc-CAP2 into HEK293 cells followed by immunoprecipitation experiments was performed. The results revealed that CAP2 interacts with CAP1. While investigating the interaction of CAP2 with other proteins, we found that ACF7, an F-actin crosslinking protein interacts with CAP2 in immunoprecipitation studies carried out on HEK293 cells, coexpressing GFP-C-ACF7 and Myc-CAP2. We further found that the domain of CAP2 interacts with myosin light chain alkali (MLC3nm) in skin lysate. In a wound healing assay CAP2 colocalised with F-actin at certain places suggesting CAP2 plays a role in wound healing. Using a conventional knock out strategy we are currently generating a mice knock out strain of CAP2 in order to learn more about the functions of this protein.

Item Type:	Thesis (PhD thesis)
Translated title:	Title Language Charakterisierung von "Cyclase Associated Protein"-Homolog, CAP2 German
Translated abstract:	Abstract Language CAP (Cyclase Associated Protein) besteht aus einer N-terminalen Domäne, die in Hefe an Adenylatzyklase binden kann, und einer C-terminalen Domäne, die für die Bindung an G-Aktin von Bedeutung ist. Beide Bereiche werden durch eine Prolin-reiche Sequenz getrennt, die, wiederum in Hefe, die Lokalisation im Zellkortex bestimmt und an SH3-Domänen binden kann. In Säugern gibt es zwei CAP Formen, CAP1 und CAP2, die durch unterschiedliche Gene kodiert werden. In dieser Arbeit wurde CAP2 untersucht. CAP2 ist im Vergleich zu CAP1 weniger stark exprimiert und zeigt eine hohe Gewebsspezifität. Es wurde nur in Gehirn, Herz, Skelettmuskel und der Haut gefunden. Seine Verteilung in diesen Organen wurde im Embryo und im adulten Organismus detailliert untersucht. Bemerkenswert ist die Verteilung im Skelettmuskel. Hier wurde eine Bänderung beobachtet wie sie für die Elemente des kontraktilen Apparates charakteristisch ist. Koimmunfärbungen mit Antikörpern gegen verschiedene Muskelproteine haben dann eine Zuordnung zur M-Bande ergeben. Eine weitere ungewöhnliche Färbung wurde beobachtet bei der Analyse der subzellulären Lokalisation. In PAM212 Zellen, einer Maus Keratinozyten Zellinie, und in primären Maus Keratinozyten ist CAP2 im Zellkern lokalisisert. Diese Lokalisierung konnte in Zellfraktionierungsexperimenten bestätigt werden. Die gleiche Verteilung wurde auch in HL-1 Zellen, einer Kardiomyozyten Zellinie, und in primären embryonalen Kardiomyozyten beobachtet. Allerdings ist die Kernlokalisation nicht in allen Zellenn zu beobachten. Die Ursache für die wechselnde Lokalisation ist nicht bekannt. CAP2 könnte auf Grund dieser Befunde zu einer neuartigen Klasse von Proteinen gehören, die zwischen Zytosol und Zellkern hin- und herwandert. Bekanntestes Beispiel ist hierfür b-Catenin. Mit der leichten Kette des Myosins und dem Protein ACF7, einem Protein, das Zytoskelettelemente verbindet und das auch im Zellkern vorkommen kann, wurden mögliche Interaktionspartner von CAP2 identifiziert. Schliesslich wude ein Vektor konstruiert, mit dem das CAP2 Gen gezielt inaktiviert werden kann und mit dessen Hilfe im weiteren Verlauf der Untersuchungen die in vivo Funktion von CAP2 geklärt werden soll. German
Creators:	Creators Email ORCID ORCID Put Code Shekar, Sunil sunil.shekar@uni-koeln.de UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-14291
Date:	2005
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Faculty of Medicine > Biochemie > Institut I für Biochemie
Subjects:	Chemistry and allied sciences
Uncontrolled Keywords:	Keywords Language Biochemie , "Cyclase Associated Protein 2" , Signaltransduktion German Biochemistry, Cyclase Associated Protein 2 , Signal transduction English
Date of oral exam:	9 February 2005
Referee:	Name Academic Title Noegel, Angelika A. Prof. Dr.
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/1429