An N-terminal Amphipathic Helix Binds Phosphoinositides and Enhances Kalirin Sec14 Domain-mediated Membrane Interactions

Abstract

Background: Promoter usage determines the peptide preceding the lipid-binding KalSec14 domain.Results: Kalirin C-promoter encodes an amphipathic helix, which interacts with phosphoinositides, localizes to the trans-Golgi network, alters KalSec14 interactions with cell membranes, and stimulates secretion. Conclusion: Sec14 domain function is altered by the preceding phosphoinositide-binding amphipathic helix. Significance: Kalirin function is expected to vary with promoter choice.Previous studies revealed an essential role for the lipid-binding Sec14 domain of kalirin (KalSec14), but its mechanism of action is not well understood. Because alternative promoter usage appends unique N-terminal peptides to the KalSec14 domain, we used biophysical, biochemical, and cell biological approaches to examine the two major products, bKalSec14 and cKalSec14. Promoter B encodes a charged, unstructured peptide, whereas promoter C encodes an amphipathic helix (Kal-Chelix). Both bKalSec14 and cKalSec14 interacted with lipids in PIP strip and liposome flotation assays, with significantly greater binding by cKalSec14 in both assays. Disruption of the hydrophobic face of the Kal-C-helix in cKalSec14 KKED eliminated its increased liposome binding. Although cKalSec14 showed significantly reduced binding to liposomes lacking phosphatidylinositol phosphates or cholesterol, liposome binding by bKalSec14 and cKalSec14 KKED was not affected. When expressed in AtT-20 cells, bKalSec14-GFP was diffusely localized, whereas cKalSec14-GFP localized to the trans-Golgi network and secretory granules. The amphipathic C-helix was sufficient for this localization. When AtT-20 cells were treated with a cell-permeant derivative of the Kal-C-helix (Kal-C-helixArg 9 ), we observed increased secretion of a product stored in mature secretory granules, with no effect on basal secretion; a cell-permeant control peptide (Kal-C-helix KKED -Arg 9 ) did not have this effect. Through its ability to control expression of a novel, phosphoinositide-binding amphipathic helix, Kalrn promoter usage is expected to affect function.Kalirin is a Rho family GDP/GTP exchange factor (GEF) 2 with widespread cell signaling functions. Alternative splicing of the Kalrn gene gives rise to multiple isoforms that are developmentally regulated and tissue-specific. Kalirin7 (Kal7), found exclusively in the nervous system, is the major isoform in the adult brain and is essential for synaptic structure and function (1-6). Kal9 and Kal12 are expressed throughout development, both within and outside of the nervous system. Kal9/12 are crucial for normal neurite outgrowth, endocrine and bone homeostasis, and smooth muscle cell migration (7-10). Kalirin proteins have multiple functional domains, with major splice variants differing at their C termini. Kal7, -9, and -12 share identical N-terminal Sec14 domains, which are followed by nine spectrin repeats and tandem Dbl and pleckstrin homology domains (GEF1), activating Rac1 and RhoG (Fig. 1A). Only Kal7 terminates in a PDZ binding motif. Kal9...

Structure and function of human Naa60 (NatF), a Golgi-localized bi-functional acetyltransferase

“…We found that residues 190–202 formed an amphipathic helix with an array of hydrophobic residues located on one side. This observation is reminiscent of the protein/membrane interaction through amphipathic helices in the cases of KalSec14, Atg3, PB1-F2 etc 20212223. In this model an amphipathic helix can immerse its hydrophobic side into the lipid bilayer through hydrophobic interactions.…”

Brain Region and Isoform-Specific Phosphorylation Alters Kalirin SH2 Domain Interaction Sites and Calpain Sensitivity

“…While Kal7 is largely particulate, much of the Kal9/Kal12 is soluble 11, 22 . Since a difference in subcellular localization could result in exposure to different protein kinases, we asked whether the extent of phosphorylation at sites common to Kal7, Kal9 and Kal12 differed.…”

“…Immunoprecipitation was carried out with slight modifications of the protocol described previously 36 using affinity-purified antibody to the Sec14 domain of Kalirin 22 For each sample, 0.8 volumes of 10% NP-40 (Pierce, SurfActs) treated with orthovanadate and PhosSTOP was added to an aliquot containing 2 mg protein; samples were allowed to rotate end over end in the cold room for 20 min and then centrifuged at 4C for 15 min at 14,000 × g to remove any insoluble material. Supernatants were then further diluted with 3.5 volumes TM-P (20 mM Na TES, 10 mM mannitol, 5 mM EDTA, 50 mM NaF, 10 mM Na pyrophosphate, pH 7.4) containing 1 mM orthovanadate, 1 tablet PhosSTOP/10 ml and Calbiochem Phosphatase Inhibitor Cocktail I.…”

“…Its Sec14 domain binds phosphoinositides and alters spine length and spine-head size 10, 1222 . Its spectrin repeat regions have many binding partners and affect spine density, spine length and synaptic size 4, 12 The PDZ-binding motif, which is unique to Kal7, interacts with PDZ domains in PSD95 and afadin, key components of the PSD 23, 24 .…”

Kalirin is required for BDNF-TrkB stimulated neurite outgrowth and branching

“…As levels of PHn-CC-EX rose, levels of Kal7 and Tiam1 C1199 proteins fell. Kal7 and Tiam1 share the ability to interact with phosphoinositides and with EphB2, NMDA receptors and spinophilin (Kiraly et al, 2011; Miller et al, 2015; Terawaki et al, 2010). The ability of PHn-CC-EX to bind phosphoinositides and multiple proteins (Joshi et al, 2013; Stam et al, 1997; Terawaki et al, 2010) may account for its ability to disrupt Rac1 activation by Kalirin.…”