Phosphorylation of multiple sites within an acidic region of Alcadein α is required for kinesin-1 association and Golgi exit of Alcadein α cargo

Abstract

Alcadein α (Alcα) is a major cargo of kinesin-1 that is subjected to anterograde transport in neuronal axons. Two tryptophan- and aspartic acid-containing (WD) motifs located in its cytoplasmic domain directly bind the tetratricopeptide repeat (TPR) motifs of the kinesin light chain (KLC), which activate kinesin-1 and recruit kinesin-1 to Alcα cargo. We found that phosphorylation of three serine residues in the acidic region located between the two WD motifs is required for interaction with KLC. Phosphorylation of these serine residues may alter the disordered structure of the acidic region to induce direct association with KLC. Replacement of these serines with Ala results in a mutant that is unable to bind kinesin-1, which impairs exit of Alcα cargo from the Golgi. Despite this deficiency, the compromised Alcα mutant was still transported, albeit improperly by vesicles following missorting of the Alcα mutant with amyloid β-protein precursor (APP) cargo. This suggests that APP partially compensates for defective Alcα in anterograde transport by providing an alternative cargo receptor for kinesin-1.

The cytoplasmic region of the amyloid β‐protein precursor (APP) is necessary and sufficient for the enhanced fast velocity of APP transport by kinesin‐1

“…Furthermore, the interaction of the WD motifs of Alca with KLC is regulated by multiple-site phosphorylation in an acidic region located between two WD motifs in the cytoplasmic region [19]. Moreover, phosphorylation of the Alca cytoplasmic region is required for the proper formation of Alca cargos by kinesin-1 [19]. These observations strongly support the functional importance of the cytoplasmic regions of cargo receptor molecules in determining the properties of vesicular cargos.…”

“…Anti‐α‐tubulin 10G10 (Wako Pure Chemicals, Ltd., Osaka, Japan), anti‐GFP (598; Medical & Biological Laboratories, Nagoya, Japan), and anti‐FLAG M2 (Sigma‐Aldrich) antibodies were purchased from the indicated suppliers. Rabbit polyclonal anti‐APP cytoplasmic antibody and anti‐Alcα cytoplasmic region antibody UT195 were described previously . HRP‐linked sheep anti‐mouse IgG and anti‐rabbit IgG antibodies were purchased from GE Healthcare Bio‐Sciences.…”

Decrease in p3‐Alcβ37 and p3‐Alcβ40, products of Alcadein β generated by γ‐secretase cleavages, in aged monkeys and patients with Alzheimer's disease

“…The Alcadein family of proteins (Alcs) comprises three members: Alcadein a (Alca), Alcadein b (Alcb), and Alcadein g (Alcg), which are type I transmembrane proteins, encoded by their respective independent genes. They are also termed calsyntenin (Clstns) exclusively expressed in neuronal tissues [1,2] and serve a variety of functions: Ca 21 -binding [3], cargo receptors for kinesin-1 [4][5][6][7][8][9], regulators of secretory pathways [10][11][12], and synaptogenesis [13][14][15].…”

Phosphorylation of KLC1 modifies interaction with JIP1 and abolishes the enhanced fast velocity of APP transport by kinesin-1

“…When KLC1 is phosphorylated at Ser460, Alcα/calsyntenin1 cargo can dissociate from kinesin-1 ( Vagnoni et al. , 2011 ), whereas multiple-site phosphorylation of Alcα is essential for the association with kinesin-1 ( Sobu et al. , 2017 ).…”

“…Phosphorylation of KLC1 at Ser460 specifically decreases binding of calsyntenin-1/Alcα ( Vagnoni et al. , 2011 ), whereas phosphorylation of Alcα at multiple seryl residues is required for its interaction with the KLC of kinesin-1 ( Sobu et al. , 2017 ).…”