Given Allison M, Ogut Ozgur, Brozovich Frank V
Division of Cardiovascular Diseases, Mayo Clinic, 200 1st Street SW, Rochester, MN 55905, USA.
Am J Physiol Cell Physiol. 2007 Jan;292(1):C432-9. doi: 10.1152/ajpcell.00175.2006. Epub 2006 Jul 26.
During nitric oxide signaling, type Ialpha cGMP-dependent protein kinase (PKGIalpha) activates myosin light chain (MLC) phosphatase through an interaction with the 130-kDa myosin targeting subunit (MYPT1), leading to dephosphorylation of 20-kDa MLC and vasodilatation. It has been suggested that the MYPT1-PKGIalpha interaction is mediated by the COOH-terminal leucine zipper (LZ) of MYPT1 and the NH(2)-terminal LZ of PKGIalpha (HK Surks and ME Mendelsohn. Cell Signal 15: 937-944, 2003; HK Surks et al. Science 286: 1583-1587, 1999), but we previously showed that PKGIalpha interacts with LZ-positive (LZ+) and LZ-negative (LZ-) MYPT1 isoforms (13). Interestingly, PKGIalpha is known to preferentially bind to RR and RK motifs (WR Dostmann et al. Proc Natl Acad Sci USA 97: 14772-14777, 2000), and there is an RK motif within the aa 888-928 sequence of MYPT1 in LZ+ and LZ- isoforms. Thus, to localize the domain of MYPT1 important for the MYPT1-PKGIalpha interaction, we designed four MYPT1 fragments that contained both the aa 888-928 sequence and the downstream LZ domain (MYPT1FL), lacked both the aa 888-928 sequence and the LZ domain (MYPT1TR), lacked only the aa 888-928 sequence (MYPT1SO), or lacked only the LZ domain (MYPT1TR2). Using coimmunoprecipitation, we found that only the fragments containing the aa 888-928 sequence (MYPT1FL and MYPT1TR2) were able to form a complex with PKGIalpha in avian smooth muscle tissue lysates. Furthermore, mutations of the RK motif at aa 916-917 (R(916)K(917)) to AA decreased binding of MYPT1 to PKGIalpha in chicken gizzard lysates; these mutations had no effect on binding in chicken aorta lysates. However, mutation of R(916)K(917) to E(916)E(917) eliminated binding, suggesting that one factor important for the PKGIalpha-MYPT1 interaction is the charge at aa 916-917. These results suggest that, during cGMP-mediated signaling, aa 888-928 of MYPT1 mediate the PKGIalpha-MYPT1 interaction.
在一氧化氮信号传导过程中，Iα型环磷酸鸟苷依赖性蛋白激酶（PKGIα）通过与130 kDa肌球蛋白靶向亚基（MYPT1）相互作用来激活肌球蛋白轻链（MLC）磷酸酶，从而导致20 kDa MLC去磷酸化并引起血管舒张。有人提出，MYPT1与PKGIα的相互作用是由MYPT1的COOH末端亮氨酸拉链（LZ）和PKGIα的NH(2)末端LZ介导的（HK Surks和ME Mendelsohn。细胞信号15: 937 - 944，2003；HK Surks等人。科学286: 1583 - 1587，1999），但我们之前表明PKGIα与LZ阳性（LZ+）和LZ阴性（LZ-）的MYPT1异构体相互作用（13）。有趣的是，已知PKGIα优先结合RR和RK基序（WR Dostmann等人。美国国家科学院院刊97: 14772 - 14777，2000），并且在LZ+和LZ-异构体的MYPT1的aa 888 - 928序列中有一个RK基序。因此，为了定位MYPT1中对MYPT1 - PKGIα相互作用重要的结构域，我们设计了四个MYPT1片段，它们既包含aa 888 - 928序列又包含下游LZ结构域（MYPT1FL），既缺乏aa 888 - 928序列又缺乏LZ结构域（MYPT1TR），仅缺乏aa 888 - 928序列（MYPT1SO），或仅缺乏LZ结构域（MYPT1TR2）。通过免疫共沉淀，我们发现只有包含aa 888 - 928序列的片段（MYPT1FL和MYPT1TR2）能够在禽平滑肌组织裂解物中与PKGIα形成复合物。此外，将aa 916 - 917处的RK基序（R(916)K(917)）突变为AA会降低鸡胗裂解物中MYPT1与PKGIα的结合；这些突变对鸡主动脉裂解物中的结合没有影响。然而，将R(916)K(917)突变为E(916)E(917)消除了结合，这表明对PKGIα - MYPT1相互作用重要的一个因素是aa 916 - 917处的电荷。这些结果表明，在cGMP介导的信号传导过程中，MYPT1的aa 888 - 928介导了PKGIα - MYPT1的相互作用。
