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. Author manuscript; available in PMC: 2008 Apr 1.
Published in final edited form as: Neuron. 2008 Jan 10;57(1):94–107. doi: 10.1016/j.neuron.2007.11.016

Figure 2.

Figure 2

CaMKI phosphorylates Ser516 in βPIX. A. In vitro phosphorylation. Purified βPIX, wild-type (WT) or the indicated mutants (S516A, 516A; T526A, 526A; S516AT526A, AA), were subjected to in vitro phosphorylation using γ[32P]ATP and purified αCaMKI (activated by βCaMKK), PKA or Pak1. Reaction products were subjected to SDS/PAGE and autoradiography. B. Phosphorylation of βPIX Ser516 by CaMKI in the isolated multiprotein signaling complex. Brain homogenates from P6 rats were immunoprecipitated with anti-βCaMKK, washed, and incubated in the presence of Mg2+/ATP without or with Ca2+/CaM, subjected SDS/PAGE and western-blotted for phospho-βPIX, GIT1, CaMKK and phospho-CaMKI (pCaMKI). C. Phosphorylation in hippocampal neurons. Cultured hippocampal neurons (7DIV) were transfected with myc-βPIX and dnCaMKK, dnCaMKI or the PKA inhibitor protein PKI and treated with 0.5 μM tetrodotoxin in ACSF for 2 hrs to suppress neuronal activity. They were then incubated for 5 min with 56 mM KCl to activate Ca2+-dependent protein kinases and PKA. Cell lysates were immunoprecipitated with myc-antibody and western-blotted for phospho-S516 βPIX. *P<0.05, **P<0.001 compared to control.