Contact

Prof. Dr. Michael Krauß
phone +49 30 94793 - 368 (lab)
krauss(at)fmp-berlin.de

Research Krauß Lab

Adaptor-dependent Cargo Sorting is Tightly Coupled to the Synthesis of Phosphoinositides

The translocation of adaptor proteins to a given membrane is initiated by a coincidence-detection mechanism that depends on the concomitant presentation of several signals in parallel, including i) membrane-resident cargo proteins , ii) distinct lipid species (frequently a PI) and iii) sometimes also an activated small GTPase like Arf. PIs, however, are not equally distributed within membranes but rather synthesized de novo at so-called ´hotspots´ (Krauss & Haucke, 2007). Thus, any coating event that depends on the presence of a certain PI must be preceded by the activation and/or recruitment of an appropriate PI-kinase. Work in our lab so far predominantly focused on endocytosis, and therefore on PI 4-phosphate 5-kinases (PIPK type I), which catalyze the major part of the PI(4,5)P2-pools found at the plasma membrane.

Interestingly, one binding partner of PIPK Iγ, the major isoform present in neurons, is the AP-2 complex itself. AP-2 associates with this enzyme independently of the presence of cargo proteins. However, incorporation of cargo-derived peptides into the complex potently stimulates the activity of the concomitantly bound kinase (Krauss et al., 2006).

 

 

 

 

 

 

Figure 1: PIPK Iγ associates with the cargo-recognizing μ2-subunit of the AP-2 complex. PIPK Iγ can therefore be purified on GST-μ2 and analyzed for enzymatic activity. PI(4,5)P2 generated in presence of 32P-ATP was detected after thin layer chromatography. In presence of AP-2μ alone PIPK Iγ displays only modest activity. However, addition of cargo-mimicking peptides (YxxΦ), but not a mutant variant (AxxΦ) exerts a potent stimulatory effect. We also analyzed a mutant form of GST-μ2 (D176A/W421A) that is incapable of associating with YxxΦ-peptides but still able to bind to PIPK Iγ. As expected, in this case peptide addition has no effect on kinase activity.

Furthermore, we identified three additional binding sites within PIPK Iγ that can associate with AP-2 (Kahlfeldt et al., 2010): Two of them contain YxxΦ-motifs and indeed bind to AP-2μ. One additional site is located within a splice insert of the kinase and forms a complex with the ear-domain of AP-2β. Based on structural analyses and affinity measurements we proposed a hierarchical model, according to which the AP-2/PIPK Iγ complex initially forms through relatively weak AP-2β-based interactions. At this initial stage the AP-2 complex is locked in an auto-inhibited conformation (conformation A in the model). Therefore, subsequent interactions with AP-2μ depend on a conformational change of the AP-2 complex, which is supported triggered by the recognition of low levels of PI(4,5)P2 at the plasma membrane (Höning et al., 2005). Cargo proteins presenting YxxΦ-motifs then associate through the respective binding pocket within AP-2μ and switch on kinase activity (conformation B). By contrast, cargo proteins lacking such motifs (for instance the synaptic vesicle protein synaptotagmin that depends on the accessory adaptor stoning 2 for tight association with AP-2) will not allow for direct activation of PIPK Iγ; in this case kinase-encoded YxxΦ-motifs may serve as a substitute (pathway B). In both pathways cargo recognition by AP-2 triggers the generation of a large pool of PI(4,5)P2 needed for the effective recruitment of coat and accessory proteins.

 

 

 

 

 

 

 

 

Figure 2: Model of PIPK Iγ activation by cargo-AP 2 interaction.

On-going research in our lab strongly suggests that other coating events at alternate subcellular localizations employ similar mechanisms to couple the membrane translocation of individual adaptor proteins with the synthesis of cognate PI species.

Leibniz-Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP)
Campus Berlin-Buch
Robert-Roessle-Str. 10
13125 Berlin, Germany
+4930 94793 - 100 
+4930 94793 - 109 (Fax)
info(at)fmp-berlin.de

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