A novel interaction of the myosin light chain phosphatase with the regulatory subunits of protein kinase A in platelets

Khalil, Jawad Shah

Abstract

Platelet activation initiates a series of events, such as shape change, degranulation and aggregation, which results in the formation of a haemostatic plug and arrest of blood loss at the sites of vascular damage. Shape change is accompanied by remodelling of the actin cytoskeleton and formation of an acto-myosin contractile ring. This process is regulated by phosphorylation of the myosin light chain (MLC), which in turn is controlled by the relative activities of myosin light chain kinase (MLCK) and phosphatase (MLCP). MLCP is a target of both inhibitory and activatory signalling molecules. Here we focussed on the control of MLCP activation by the cyclic adenosine 3', 5’ monophosphate (cAMP) signalling pathway. cAMP signalling is a major inhibitory pathway that inhibits platelet function through its effector protein kinase A (PKA). One of the downstream targets of PKA is MLCP. In vascular smooth muscle cells cAMP activates MLCP to dephosphorylate MLC and regulate cytoskeletal rearrangement. However, the relationship between cAMP signalling, MLCP and platelet function is unclear. Here we investigated the molecular and biochemical regulation of MLCP by cAMP signalling.

We found that MYPT1, the targeting subunit of MLCP, is phosphorylated in platelets in response to cAMP elevating agents, suggesting that the phosphatase is a direct target for cAMP signalling. Our data suggests the possibility of at least two different splice variants of MYPT1 in platelets, but only one splice variant, possibly full length, was phosphorylated downstream of cAMP signalling. Using co-immunoprecipitation, cAMP pull-down assays and GST pull-down approaches we found that MYPT1 and PKA are part of the same complex and MYPT1 interacts with all four regulatory subunits of PKA (PKA-R) in platelets. Using a series of truncated proteins in HEK cells the interaction of MYPT1 and PKA-R was mapped to the central region of MYPT1 (aa501-706). Moreover, co-sedimentation assays with recombinant proteins confirmed the direct association of MYPT1 (aa501-706) with PKA-R. The conserved dimerisation and docking (D/D) domain of PKA-R, which facilitates interactions with A-kinase anchoring proteins (AKAP) was not required for the interaction. Consistent with this observation, the AKAP disruptor peptide Ht31 did not disrupt the interaction, indicating that the interaction of MYPT1 with PKA-R does not occur in an AKAP modus.

These results were complemented with immunohistochemistry studies showing that MYPT1 co-localised with all four PKA regulatory subunits in both non-activated and spread platelets. In summary, our data identify MYPT1 as a novel PKA binding protein in platelets. The interaction of MYPT1 with PKA emerges as an important component of the signalling pathways that protect platelets from a hyperreactive state and may constitute a target towards preventing thrombogenic disorders.

Citation

Khalil, J. S. A novel interaction of the myosin light chain phosphatase with the regulatory subunits of protein kinase A in platelets. (Thesis). Hull York Medical School, the University of Hull and the University of York. https://hull-repository.worktribe.com/output/4221504

• Thesis Type: Thesis
• Deposit Date: Apr 29, 2019
• Publicly Available Date: Feb 23, 2023
• Keywords: Medicine
• Public URL: https://hull-repository.worktribe.com/output/4221504
• Additional Information: Hull York Medical School, The University of Hull and The University of York
• Award Date: May 1, 2018