The effector domain of human Dlg tumor suppressor acts as a switch that relieves autoinhibition of kinesin-3 motor GAKIN/KIF13B - PubMed (original) (raw)

. 2007 Sep 4;46(35):10039-45.

doi: 10.1021/bi701169w. Epub 2007 Aug 14.

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The effector domain of human Dlg tumor suppressor acts as a switch that relieves autoinhibition of kinesin-3 motor GAKIN/KIF13B

Kaori H Yamada et al. Biochemistry. 2007.

Abstract

The activity of motor proteins must be tightly regulated in the cells to prevent unnecessary energy consumption and to maintain proper distribution of cellular components. Loading of the cargo molecule is one likely mechanism to activate an inactive motor. Here, we report that the activity of the kinesin-3 motor protein, GAKIN, is regulated by the direct binding of its protein cargo, human discs large (hDlg) tumor suppressor. Recombinant GAKIN exhibits potent microtubule gliding activity but has little microtubule-stimulated ATPase activity in solution, suggesting that it exists in an autoinhibitory form. In vitro binding measurements revealed that defined segments of GAKIN, particularly the MAGUK binding stalk (MBS) domain and the motor domain, mediate intramolecular interactions to confer globular protein conformation. Direct binding of the SH3-I3-GUK module of hDlg to the MBS domain of GAKIN activates the microtubule-stimulated ATPase activity of GAKIN by approximately 10-fold. We propose that the cargo-mediated regulation of motor activity constitutes a general paradigm for the activation of kinesins.

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Figures

FIGURE 1

FIGURE 1

Autoinhibition of full length GAKIN protein. (A) Expression and purification of GAKIN. Motor-FHA and Full length (FL)-GAKIN recombinant proteins were expressed in insect cells and purified with Ni++ affinity purification. CBB stained 8% SDS-PAGE gel is shown. (B) Microtubule stimulated ATPase activity of recombinant GAKIN proteins. Motor-FHA and/or FL-GAKIN were incubated with or without 2.5 μM of microtubules and 1.0 mM MgATP. ATPase activity was determined by measuring phosphate release by the malachite green method. (C) Microtubule stimulated ATPase activity of motor-FHA and FL-GAKIN as a function of microtubule concentration. (D) Chemical cross-linking of full length GAKIN. Full length GAKIN was incubated with or without cross-linker and separated in 4% SDS-PAGE gel. (E) Microtubule gliding activity of FL-GAKIN. Movement of the fluorescent labeled Taxol-stabilized microtubules was recorded and their velocity distribution is shown in the histogram. Scale bar represents 5 μm.

FIGURE 2

FIGURE 2

Intramolecular interactions mediated by MBS domain of GAKIN. (A) Schematic representation of GAKIN truncated constructs used for the binding assay. (B) Binding of stalk1 region of GAKIN with motor and CT. 35S labeled stalk1 was expressed in the rabbit reticulocyte lysate in vitro translation system (STP3, Novagen), and S-tag pull down was performed with S-motor1 or S-CT immobilized on S-protein agarose beads or plain beads as a control. Bound 35S-labeled stalk1 protein was detected by fluorography. (C) Pull down assay from 293T cell lysate. GFP-fused motor2 and motor-FHA were transiently expressed in 293T cells, and S-tag pull down was performed with control S, S-motor1, S-FHA, S-stalk2 and S-CT. Bound proteins were detected by Western blot using anti-GFP antibody. The bottom panel shows the CBB stained gel of the S-fusion protein used for binding. (D) Direct binding of the GST-motor2 and GST-GUK (hDlg) with Trx-fusion proteins of GAKIN. Purified recombinant proteins, GST-motor2 and GST-GUK (hDlg) were used for direct binding with S, S-stalk2, S-MBS and S-stalk3, immobilized on S-protein agarose. Bound proteins were detected by anti-GST Western blot. (E) Direct binding of GST-CT with S-stalk2.

FIGURE 3

FIGURE 3

Activation of GAKIN by direct interaction of hDlg. (A) Schematic representation of GST fused hDlg constructs used in the study. (B) Binding of GST-PIP3BP and GST-hDlg proteins with GAKIN. Purified FL-GAKIN was used for direct binding with GST-fusion proteins immobilized on the beads. GAKIN was detected with Western blot using anti-GAKIN pAb (10). In the bottom panel, GST-fusion proteins were shown in the CBB stained gel. (C) Microtubule stimulated ATPase activity of FL-GAKIN in the presence of GST-fusion proteins. GST-fusion proteins of PIP3BP and hDlg (1 μM, except for FL-I3 and FL-I3-L613P, which were 0.5 μM) mixed with FL-GAKIN (50 nM) were incubated with or without microtubules (1 μM). (D) Microtubule dependent ATPase activity as a function of SH3-I3-GUK concentration. FL-GAKIN (50 nM) and microtubules (1 μM) were used in the reaction. (E) Microtubule concentration dependent ATPase activity of FL-GAKIN with and without SH3-I3-GUK. FL-GAKIN (50 nM) and GST-SH3-I3-GUK of hDlg (1 μM) were used in the reaction.

FIGURE 4

FIGURE 4

Proposed model of the regulation of GAKIN by hDlg binding. GAKIN forms intramolecular interactions and is inactive in solution in vitro. Binding of SH3-I3-GUK of hDlg relieves the intramolecular inhibition of GAKIN and frees its motor domain accessible to microtubules. Because the binding of full length hDlg is not sufficient to activate GAKIN, some as yet unknown mechanism must alter the conformation of full length hDlg so that it can activate GAKIN in vivo. Stable interactions of hDlg with proteins residing on the membrane vesicles might trigger the conformational change of hDlg, which in turn activate GAKIN, thereby initiating the trafficking of the vesicles along microtubules.

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