human pak1  (Cytoskeleton Inc)

Journal: JCI Insight

Article Title: Rac1 palmitoylation is required for cardiac stress adaptation and regulation of protein kinase A signaling

doi: 10.1172/jci.insight.193733

Figure Lengend Snippet: Circulating levels of norepinephrine and angiotensin II are elevated during chronic hypertrophic stress. Activation of the β 1 AR elicits G s -dependent activation of adenylyl cyclase, leading to cAMP production and protein kinase A (PKA) activation. Active PKA phosphorylates substrates critical for excitation-contraction coupling. AT1R activation leads to Rac1 activation and promotes Rac1 S -palmitoylation cycling. S -palmitoylated Rac1 is required to evoke dephosphorylation of PKA substrates and dampen excessive adrenergic signaling that promotes cardiac maladaptation, likely through Pak1-dependent activation of PP2A. Upon loss of Rac1 palmitoylation cycling at Cys-178 (as observed in Rac1 cKI hearts), PP2A-mediated antagonism of PKA substrate activity is not properly regulated, resulting in hyperphosphorylation of PKA substrates that chronically promotes cardiac decompensation, systolic dysfunction, and adverse hypertrophic remodeling. AC, adenylyl cyclase; AngII, angiotensin II; AT1R, angiotensin II receptor type I; β 1 AR, β1 adrenergic receptor; LTCC, L-type calcium channel; MyBPC3, myosin binding protein C3; NE, norepinephrine; Pak1, p21-activated kinase-1; PKA, protein kinase A; PLN, phospholamban; PP2A, protein phosphatase 2A; SERCA, sarco-endoplasmic reticulum calcium ATPase; TnI, troponin I; TnT, troponin T.

Article Snippet: For experiments in NRCMs with overexpressed Rac1, we generated a recombinant glutathione S -transferase (GST)-PAK-PBD fusion protein by transforming BL21-DE3 E . coli with a pGEXT-PAK-PBD-70-117 plasmid that contains residues 70–117 of human Pak1 (Addgene, 12217, gift from Jonathan Chernoff).

Techniques: Activation Assay, De-Phosphorylation Assay, Activity Assay, Binding Assay