Characterization of the CHL1 – hsc70 interaction

VI. Discussion

elements, it was suggested that the heat shock cognate may regulate those filamentous reorganizations during early mouse brain development.

As mentioned before, the conformational alterations of substrate proteins generated by hsc70 can regulate signal transduction pathways. The involvement of hsc70 in certain signalling cascades was intensively investigated regarding the interaction of hsc70 with the anti-apoptotic protein Bag-1 (Takayama et al., 1997). It was shown that the formation of the hsc70/Bag-1 complex can regulate the activity of raf-1 by competetive binding of this serine/threonine kinase and subsequently the activity of the downstream extracellular signal-regulated kinases erk1 and erk2 is affected (Song et al., 2001). The hsc70 binding protein Bag-1 was also described to interact with the adaptor protein 14-3-3 that modulates signalling events between components of different pathways (Fountoulakis et al., 1999). Disruption of the 14-3-3 function revealed an impaired activation of the erk kinases but increases basal activation levels of the JNK1 kinase and the stress kinase p38 (Xing et al., 2000) suggesting a further signalling pathway that might be influenced by hsc70 in cooperation with Bag-1 and 14-3-3.

VI. Discussion

Furthermore, the co-immunoprecipitation of hsc70 was additionally carried out by the use of a polyclonal anti L1 antiserum. Neither using L1-ICD nor using the intracellular domain of NCAM180 in the overlay assay and also not after immunoprecipitation of L1, an interaction with hsc70 was observed. A sequence analysis of CHL1 revealed the presence of the HPD tripeptide that is a highly conserved motif exposed in the J-domain of molecular chaperones within the intracellular portion of CHL1. This putative binding motif was identified to be the essential structure for the binding of CHL1 to hsc70 using a site-directed mutagenesis that led to a single amino acid exchange of the histidine or the aspertate residue. Deletion of only one amino acid in the HPD tripeptide resulted in a complete loss of hsc70 binding by mutant CHL1. An alignment of the intracellular domains of several L1-related molecules showed that the HPD binding motif is exclusively present in the sequence of CHL1 and not in the sequences of L1, NrCAM, NgCAM, neurofascin, neuroglian or the zebrafish L1-homologues L1.1 and L1.2 (Holm et al., 1996) suggesting that the interaction between a neuronal cell adhesion molecule and the heat shock cognate is an exclusive property of CHL1.

Furthermore, the interaction of CHL1 and hsc70 was identified to be strictly ADP-dependent since no co-immunoprecipitation of hsc70 and CHL1 was observed in the presence of ATP but in the presence of ADP. Hsc70 includes an ATPase function and binding of either ADP or ATP was previously described as a functional feature of hsc70 for the recruitment of substrates or further co-chaperones that can influence the binding properties of the heat shock cognate.

Although the conditions prevailing during the binding assay that was used for the isolation of the CHL1 binding protein were rather denaturing, a strong and direct binding between CHL1 and hsc70 was observed in this approach. Interestingly, the direct binding under native conditions using recombinant fusion proteins failed to confirm the CHL1 – hsc70 association whereas the co-localization of both proteins that was observed in a distinct distribution pattern in hippocampal neurons was presumed as an indication for the binding between these proteins. The interaction was confirmed in an alternative binding approach using a co-precipitation of CHL1 and hsc70 from a crude cell lysates. Finally, the interaction of CHL1 and hsc70 was shown to be restricted to a distinct detergent-insoluble membrane subdomain that was assumingly a so-called raft fraction. Taking these results together, a specific conformation of CHL1 was proposed to be required for the binding to hsc70 that was mimicked by the denaturing conditions prevailing during the electrophoretic separation.

VI. Discussion

Interestingly, such a particular binding assay was used to verify the interaction of hsc70 with another binding partner indicating that the electrophoretic separation under denaturing conditions did not interfere with the binding properties of hsc70. The interaction between hsc70 and the anti-apoptotic protein Bag-1 was also revealed by this approach using ligand blotting of hsc70 and subsequent incubation of purified Bag-1 as a soluble probe (Höhfeld, 1998). The ELISA binding assay that was carried out in the present study to confirm the interaction of hsc70 and CHL1 also supported the assumption that the conformational status of the interaction partners might be a crucial factor for the binding. Recombinant Bag-1 and hsc70 were applied as a positive control. An interaction between these known binding partners was only seen when hsc70 was immobilized and Bag-1 was incubated as a soluble protein, whereas after immobilization of Bag-1 and subsequent incubation of soluble hsc70, no interaction was detectable. This result suggests that on one hand a distinct three-dimensional conformation of Bag-1 is an important prerequisite for the binding of hsc70 that is altered after the immobilization to the plastic surface (personal communication, Dr. U.

Hartl, Munich). On the other hand, the immobilization of hsc70 also modifies the conformation of the protein without disturbing the binding to Bag-1 indicating that the immobilization even may contribute to a specific conformation of hsc70 that first provides the binding as it was previously seen in the overlay approach. In the cellular environment, this specific conformation of hsc70 is thought to be generated and also regulated by assisting proteins which may be co-chaperones of the hsc70. This presumption is supported by the immunocytochemical detection of hsc70 in primary hippocampal neurons revealing a distinct distribution pattern of the cytoplasmic protein in cellular subdomains. The co-localization of hsc70 and CHL1 was also detectable in specific areas indicating that the binding might be facilitated only in those distinct subdomains. The distribution analysis of hsc70 and CHL1 in membrane subfractions separated from an enriched synaptosomal preparation further supported the assumption that the binding of hsc70 and CHL1 may occur only in restricted subdomains since both proteins showed an overlapping distibution pattern in all subfractions which were analyzed. Finally, a co-immunoprecipitation of hsc70 and CHL1 that was exclusively observed in crude membranes as well as in the detergent-insoluble fraction of brain membranes confirmed the observation that the interaction of hsc70 and CHL1 is restricted to cellular subcompartment. The localization of the CHL1 – hsc70 interaction to subdomains may indicate that further proteins assist the interaction since in the non-detergent

VI. Discussion

soluble as well as in the Triton-soluble fraction both binding partners were present but no interaction was detectable, whereas in the detergent-insoluble fraction the expression of CHL1 was lower when compared to the soluble fractions but the co-precipitation was remarkably pronounced. An identification of such assisting proteins would be of outstanding interest for the further understanding by which mechansims a complex formation containing hsc70 can promote and/or regulate the in vivo binding of CHL1 and hsc70.

Palmityolation of serine residues that can influence the translocation of a fluid membrane protein to rafts (Kabouridis et al., 1997; Zhang et al., 1998) was addressed to be involved in the localization of CHL1 in cellular subdomains. A serine residue is not present in the intracellular domain of CHL1 but is localized immediately beyond the cytoplasmic part at position three within the transmembrane portion. This particular serine residue was affected by a site-directed mutagenesis. A decreased translocation of CHL1 to the detergent insoluble fraction was assumed that may reduce the facilitation of the CHL1 – hsc70 interaction since decreased levels of CHL1 should be present in the Triton X-insoluble fraction. Although CHL1 was only weakly detectable in the detergent insoluble fraction of CHO cell lysates and co-immunoprecipitations of CHL1 and hsc70 were performed using a Triton X-100 soluble fraction, the effect of the serine residue exchange was revealed indicated by a slightly reduced co-precipitation of hsc70. A decrease in the co-precipitation of hsc70 and the mutant affecting palmityolation was seen in CHO cells as well as in N2A cells. In both cellular systems, the co-precipitation was carried out using either a detergent soluble or a crude lysate indicating that the localization of the binding partners to detergent insoluble subdomains is no prerequisite for the interaction of CHL1 and hsc70 in those particular cells. Nevertheless, the effect of the serine mutation that presumably reduces the translocation of CHL1 to rafts suggest that the interaction is also facilitated by a localization of CHL1 in the detergent insoluble fraction.

The conformation of the binding partners and thereby the translocation of both proteins to cellular subdomains obviously is a crucial factor for the facilitation of the CHL1 – hsc70 interaction. In addition to this, the immunocytochemical analysis of hippocampal cells demonstrated that the interaction between CHL1 and hsc70 depends on the developmental stage of the cells since a co-localization of CHL1 and hsc70 in distinct areas was only observed in hippocampal neurons derived from 4-day old animals and after culturing for three days, whereas in cells prepared from newborn animals cultured for only one day,

co-VI. Discussion

localization was only seen after co-capping of CHL1. This assumption was further investigated by biochemical analysis performing a co-immunoprecipitation of CHL1 and hsc70 from membrane fractions which were derived from either 5-day old or 3-weeks old mice. It was shown that the co-precipitation using crude membranes was much more pronounced in older animals when compared to younger animals although the expression levels of CHL1 were remarkably higher in the 5-day old mice. The crude membranes were further separated in detergent-soluble and insoluble fractions and the subsequent immunoprecipitation of CHL1 revealed a co-precipitation of hsc70 only in the detergent-insoluble fraction that was prepared from 3-weeks old animals. The co-immunoprecipitation using membrane fractions derived from animals of different ages confirmed the assumption that not only the conformation of the binding partners but also the developmental stage of the animals facilitate the interaction of CHL1 and hsc70. As mentioned before, hsc70 shows an elevated expression during early neuronal development, whereas CHL1 becomes weakly detectable at embryonic day 13 and shows the highest expression levels at rather late developmental stages from embryonic day 18 to postnatal day 7 (Hillenbrand et al., 1999).

Furthermore, the biochemical analysis of the CHL1 – hsc70 binding that was carried out with regard to the age of the animals indicated that the interaction between both proteins was more pronounced in older animals than in 5-day old animals. Taken these observations together, a hsc70 function during early developmental stages in mice without an involvement of CHL1 is assumed whereas a functional interaction of CHL1 and hsc70 is facilitated at later developmental stages including the postnatal development in up to 3-weeks old animals and probably also in adults. Thereby, a possible function of the CHL1 – hsc70 interaction is proposed suggesting that the binding of both proteins at later developmental stages may confers stability and/or maintainance of cellular structures to neurons whereas during early development of the mouse brain, hsc70 is probably more involved in alterations of structural elements including conformational changes of cytoskeletal components.