而且，當ZAK 和RhoGDIβ同時表達時，會抑制ZAK 所調控ANF 的表達 。以ZAK specific siRNA knockdown ZAK 的表達，可恢復 RhoGDIβ的活性。RhoGDIβ在H9c2 心肌細胞藉由調控Rac1 表達，來引發細胞的肥大性生長；而且RhoGDIβ調控的細胞移動呈現Rac1 依賴。以siRNA knockdown RhoGDIβ的過度表達時，可降低H9c2 心肌細胞的移動。然而RhoGDIβ所引發H9c2 心肌細胞的移動受到ZAK的負向調控，與細胞的增殖(cell proliferation)並無關聯。
ZAK belongs to the mixed lineage kinases, a family of serine/threonine kinases that are classified as MAP3Ks and can activate the JNK and nuclear factor κB (NFκB) pathway ZAK induces JNK activation through a dual phosphorylation kinase, JNKK2/MKK7.
To identify effectors of ZAK and to study the ZAK signaling cascade,we used a yeast two-hybrid system to isolate ZAK effectors from a human heart cDNA library. One of the isolated cDNAs encoded Rho GDP dissociation inhibitor beta (RhoGDIβ).However, only their ability to sequester RhoGTPases is well understood. Therefore, the question
remains as to whether RhoGDIβ is actually a RhoGTPase regulator.
Overexpression of RhoGDIβ, a Rho GDP dissociation inhibitor, induced hypertrophic growth and suppressed cell cycle progression in a cultured cardiomyoblast cell line. Knockdown of RhoGDIβ expression by RNA interference blocked hypertrophic growth. The further studies demonstrated that RhoGDIβ physically interacts with ZAK and is phosphorylated by ZAK in vitro, and this phosphorylation negatively regulates RhoGDIβ functions.
Moreover, the ZAK-RhoGDIβ interaction may maintain ZAK in an inactive hypophosphorylated form. These two proteins could negatively regulate one another such that ZAK suppresses RhoGDIβ functions through phosphorylation and RhoGDIβ counteracts the effects of ZAK by physical
interaction.
Knockdown of ZAK expression in ZAK- and RhoGDIβ-expressing cells by ZAK-specific RNA interference restored the full functions of RhoGDIβ. At our previous study demonstrated that RhoGDIβ plays a undefined role in regulating Rac1 expression through transcription to induce hypertrophic growth and cell migration and that these functions are
blocked by the expression of a dominant-negative form of Rac1.Knockdown
of RhoGDIβ expression by RNA interference blocked RhoGDIβ-induced Rac1 expression and cell migration.
We demonstrated that the co-expression of ZAK and RhoGDIβ in cells resulted in an inhibition in the activity of ZAK to induce ANF expression. Knockdown of ZAK expression in ZAK-RhoGDI β-expressing cells by ZAK-specific RNA interference restored the activities of RhoGDIβ.
Expression of RhoGDIβ induces hypertrophic growth via
modulation of Rac1 expression in H9c2 cardiac cells. H9c2 cell migration promoted by RhoGDIβ is Rac1 dependent . Knockdown of overexpressed RhoGDIβby siRNA reduces H9c2 cell migration. RhoGDIβ-induced cell migration does not correlate with cell proliferation ，and RhoGDIβ
-induced wound healing is negatively regulated by ZAK.
