Elevated Expression of the Integrin-Associated Protein PINCH Suppresses the Defects of Drosophila melanogaster Muscle Hypercontraction Mutants
Figure 3
PINCHD303V mutation disrupts binding to RSU1.
A) Schematic of PINCH showing its 5 LIM domain structure. The N-terminal ankyrin repeat of ILK binds to LIM1 of PINCH via PINCHQ38. Parvin associates with PINCH indirectly via binding to the ILK pseudokinase domain. RSU1 binds to LIM5 of PINCH through PINCHD303. B) Sequence alignment of the C-terminus of fly, mouse and human PINCH sequences demonstrates the conservation of D303 (boxed with arrowhead). Asterisks denote invariant residues, and colons denote conserved residues. C) Sequence alignment using the 5 individual LIM domains of Drosophila PINCH shows that D303 (boxed with arrowhead) is a variable residue within LIM sequences. Dots denote the zinc binding residues. D) Ni-NTA pull-downs of wild type PINCH-His and PINCHD303VHis expressed in S2 cells show a disruption of RSU1 binding only in the D303V mutant while ILK binding is preserved. E) Flag immunoprecipitations of adult fly lysates from PINCH null mutants rescued with either wild type PINCH-Flag or PINCHD303VFlag transgenes. w1118 is used as a negative control that does not express Flag. ILK, RSU1 and a PINCH transgene are all expressed in the starting material. PINCHD303VFlag pulls down ILK but fails to co-precipitate RSU1. In both D and E, densitometric analyses were conducted to compare levels of protein between samples and quantification is provided below each blot. α-tubulin was used as a loading control and values for PINCH, RSU1 and ILK were adjusted to account for variation in loading.
