Abstract
Mutations that reduce expression or give rise to a Thr85Ser (T85S) mutation of Ca2+-CaM-dependent protein kinase kinase-2 (CaMKK2) have been implicated in behavioural disorders such as anxiety, bipolar and schizophrenia in humans. Here we report that Thr85 is an autophosphorylation site that endows CaMKK2 with a molecular memory that enables sustained autonomous activation following an initial, transient Ca2+ signal. Conversely, autophosphorylation of Ser85 in the T85S mutant fails to generate autonomous activity but instead causes a partial loss of CaMKK2 activity. The loss of autonomous activity in the mutant can be rescued by blocking glycogen synthase kinase-3 (GSK3) phosphorylation of CaMKK2 with the anti-mania drug lithium. Furthermore, CaMKK2 null mice representing a loss of function model the human behavioural phenotypes, displaying anxiety and manic-like behavioural disturbances. Our data provide a novel insight into CaMKK2 regulation and its perturbation by a mutation associated with behavioural disorders.
Highlights
Thought to occlude the active site in the absence of Ca2+-CaM; i.e. by an intrasteric autoinhibition mechanism that blocks access of substrates[14]
Prompted by the human genetic association studies we have determined the functional consequences of the T85S mutation on human calmodulin dependent protein kinase kinase-2 (CaMKK2) activity and investigated the effect of CaMKK2 deletion on behavior in mice
The level of autonomous activity generated by autophosphorylation is similar to the maximal activation achieved with 10 μ M Ca2+, but ~50% less than with 50 μ M Ca2+
Summary
Using purified enzyme and pThr[85] or pSer[85] phosphospecific antibodies, we found both Thr[85] in human WT CaMKK2 and Ser[85] in the T85S mutant underwent autophosphorylation in the presence of Ca2+-CaM and MgATP, but not when incubated with MgATP alone or in a kinase inactive mutant (D312A) of CaMKK2 (Fig. 1a; Supplementary Fig. 1a,b; Supplementary Fig. 2). Our data show that human CaMKK2 has two distinct mechanisms to achieve autonomous activity i.e Thr[85] autophosphorylation or Ser129/Ser[133] dephosphorylation (Fig. 2b). Multiple convergent lines of evidence point strongly to reductions in CaMKK2 expression/ activity with behavioural disorders: 1) Our results show that CaMKK2 null mice exhibit behavioural disturbances similar to those observed in humans with behavioural disorders; 2) We show the T85S mutant linked with bipolar and anxiety in humans is unable to generate autonomous activity via Thr[85] autophosphorylation and causes a intermittent loss of activity; 3) Reduced expression of CaMKK2 is associated with schizophrenia in seven diverse human populations2; 4) Mice treated with methamphetamine to induce bipolar-like symptoms have decreased CaMKK2 expression, but normal expression is maintained with co-treatment of the mood stabiliser valproate[3]. Our data provide a mechanistic insight into the regulation of CaMKK2 activity and its perturbation by a mutation associated with behavioural disorders
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